Maize inbred PH4D3N

ABSTRACT

A novel maize variety designated PH4D3N and seed, plants and plant parts thereof are provided. Methods for producing a maize plant comprise crossing maize variety PH4D3N with another maize plant are provided. Methods for producing a maize plant containing in its genetic material one or more traits introgressed into PH4D3N through backcross conversion and/or transformation, and to the maize seed, plant and plant part produced thereby are provided. Hybrid maize seed, plants or plant parts are produced by crossing the variety PH4D3N or a locus conversion of PH4D3N with another maize variety.

BACKGROUND

There are numerous steps in the development of any novel, desirablemaize variety. Plant breeding begins with the analysis and definition ofproblems and weaknesses of the current germ plasm, the establishment ofprogram goals, and the definition of specific breeding objectives. Thenext step is selection of germplasm that possess the traits to meet theprogram goals. The breeder's goal is to combine in a single variety orhybrid, various desirable traits. For field crops, these traits mayinclude resistance to diseases and insects, resistance to heat anddrought, reducing the time to crop maturity, greater yield, alteredfatty acid profile, abiotic stress tolerance, improvements incompositional traits, and better agronomic characteristics and quality.

These product development processes, which lead to the final step ofmarketing and distribution, can take from six to twelve years from thetime the first cross is made until the finished seed is delivered to thefarmer for planting. Therefore, development of new varieties and hybridsis a time-consuming process. A continuing goal of maize breeders is todevelop stable, high yielding maize varieties and hybrids that areagronomically sound with maximal yield over one or more differentconditions and environments.

SUMMARY

Provided is a novel maize, Zea mays L., variety, designated PH4D3N andprocesses for making PH4D3N. Seed of maize variety PH4D3N, plants ofmaize variety PH4D3N, plant parts and cells of maize variety PH4D3N, andto processes for making a maize plant that comprise crossing maizevariety PH4D3N with another maize plant are provided. Also provided aremaize plants having all the physiological and morphologicalcharacteristics of the inbred maize variety PH4D3N.

Processes are provided for making a maize plant containing in itsgenetic material one or more traits introgressed into PH4D3N through oneor more of backcross conversion, genetic manipulation andtransformation, and to the maize seed, plant and plant parts producedthereby. Hybrid maize seed, plants or plant parts produced by crossingthe variety PH4D3N or a locus conversion of PH4D3N with another maizevariety are also provided.

The inbred maize plant may further comprise a cytoplasmic or nuclearfactor capable of conferring male sterility or otherwise preventingself-pollination, such as by self-incompatibility. Parts of the maizeplant described herein are also provided, for example, pollen obtainedfrom an inbred plant and an ovule of the inbred plant.

Seed of the inbred maize variety PH4D3N is provided. The inbred maizeseed may be an essentially homogeneous population of inbred maize seedof the variety designated PH4D3N. Essentially homogeneous populations ofinbred seed are generally free from substantial numbers of other seed.Therefore, inbred seed generally forms at least about 97% of the totalseed. The population of inbred maize seed may be particularly defined asbeing essentially free from hybrid seed. The inbred seed population maybe separately grown to provide an essentially homogeneous population ofinbred maize plants designated PH4D3N.

Compositions are provided comprising a seed of maize variety PH4D3Ncomprised in plant seed growth media. In certain embodiments, the plantseed growth media is a soil or synthetic cultivation medium. In specificembodiments, the growth medium may be comprised in a container or may,for example, be soil in a field.

Maize variety PH4D3N comprising an added heritable trait is provided.The heritable trait may comprise a genetic locus that is a dominant orrecessive allele. In certain embodiments, a plant of maize varietyPH4D3N comprising a single locus conversion is provided. The locusconversion may be one which confers one or more traits such as, forexample, male sterility, herbicide tolerance, insect resistance, diseaseresistance (including, for example) bacterial, fungal, nematode or viraldisease, waxy starch, modified fatty acid metabolism, modified phyticacid metabolism, modified carbohydrate metabolism and modified proteinmetabolism is provided. The trait may be, for example, conferred by anaturally occurring maize gene introduced into the genome of the varietyby backcrossing, a natural or induced mutation, or a transgeneintroduced through genetic transformation techniques. When introducedthrough transformation, a genetic locus may comprise one or moretransgenes integrated at a single chromosomal location.

An inbred maize plant of the variety designated PH4D3N is provided,wherein a cytoplasmically-inherited trait has been introduced into theinbred plant. Such cytoplasmically-inherited traits are passed toprogeny through the female parent in a particular cross. An exemplarycytoplasmically-inherited trait is the male sterility trait.Cytoplasmic-male sterility (CMS) is a pollen abortion phenomenondetermined by the interaction between the genes in the cytoplasm and thenucleus. Alteration in the mitochondrial genome and the lack of restorergenes in the nucleus will lead to pollen abortion. With either a normalcytoplasm or the presence of restorer gene(s) in the nucleus, the plantwill produce pollen normally. A CMS plant can be pollinated by amaintainer version of the same variety, which has a normal cytoplasm butlacks the restorer gene(s) in the nucleus, and continues to be malesterile in the next generation. The male fertility of a CMS plant can berestored by a restorer version of the same variety, which has therestorer gene(s) in the nucleus. With the restorer gene(s) in thenucleus, the offspring of the male-sterile plant can produce normalpollen grains and propagate. A cytoplasmically inherited trait may be anaturally occurring maize trait or a trait introduced through genetictransformation techniques.

A tissue culture of regenerable cells of a plant of variety PH4D3N isprovided. The tissue culture can be capable of regenerating plantscapable of expressing all of the physiological and morphological orphenotypic characteristics of the variety, and of regenerating plantshaving substantially the same genotype as other plants of the variety.Examples of some of the physiological and morphological characteristicsthat may be assessed include characteristics related to yield, maturity,and kernel quality. The regenerable cells in such tissue cultures can bederived, for example, from embryos, meristematic cells, immaturetassels, microspores, pollen, leaves, anthers, roots, root tips, silk,flowers, kernels, ears, cobs, husks, or stalks, or from callus orprotoplasts derived from those tissues. Maize plants regenerated fromthe tissue cultures, and plants having all the physiological andmorphological characteristics of variety PH4D3N are also provided.

Processes are provided for producing maize seeds or plants, whichprocesses generally comprise crossing a first parent maize plant as amale or female parent with a second parent maize plant, wherein at leastone of the first or second parent maize plants is a plant of the varietydesignated PH4D3N. These processes may be further exemplified asprocesses for preparing hybrid maize seed or plants, wherein a firstinbred maize plant is crossed with a second maize plant of a different,distinct variety to provide a hybrid that has, as one of its parents,the inbred maize plant variety PH4D3N. In these processes, crossing willresult in the production of seed. The seed production occurs regardlessof whether the seed is collected or not.

In some embodiments, the first step in “crossing” comprises planting,such as in pollinating proximity, seeds of a first and second parentmaize plant, and preferably, seeds of a first inbred maize plant and asecond, distinct inbred maize plant. Where the plants are not inpollinating proximity, pollination can be achieved by transferring apollen or tassel bag from one plant to the other as described below.

A second step comprises cultivating or growing the seeds of said firstand second parent maize plants into plants that bear flowers-maleflowers (tassels) and female flowers (silks).

A third step comprises preventing self-pollination of the plants, i.e.,preventing the silks of a plant from being fertilized by any plant ofthe same variety, including the same plant. This can be done byemasculating the male flowers of the first or second parent maize plant,(i.e., treating or manipulating the tassels so as to prevent pollenproduction, to produce an emasculated parent maize plant).Self-incompatibility systems may also be used in some hybrid crops forthe same purpose. Self-incompatible plants still shed viable pollen andcan pollinate plants of other varieties but are incapable of pollinatingthemselves or other plants of the same variety.

A fourth step may comprise allowing cross-pollination to occur betweenthe first and second parent maize plants. When the plants are not inpollinating proximity, this is done by placing a bag, usually paper orglassine, over the tassels of the first plant and another bag over thesilks of the incipient ear on the second plant. The bags are left inplace for at least 24 hours. Since pollen is viable for less than 24hours, this assures that the silks are not pollinated from other pollensources, that any stray pollen on the tassels of the first plant isdead, and that the only pollen transferred comes from the first plant.The pollen bag over the tassel of the first plant is then shakenvigorously to enhance release of pollen from the tassels, and the shootbag is removed from the silks of the incipient ear on the second plant.Finally, the pollen bag is removed from the tassel of the first plantand is placed over the silks of the incipient ear of the second plant,shaken again and left in place. Yet another step comprises harvestingthe seeds from at least one of the parent maize plants. The harvestedseed can be grown to produce a maize plant or hybrid maize plant.

Also provided are maize seed and plants produced by a process thatcomprises crossing a first parent maize plant with a second parent maizeplant, wherein at least one of the first or second parent maize plantsis a plant of the variety designated PH4D3N. In one embodiment, maizeseed and plants produced by the process are first generation (F1) hybridmaize seed and plants produced by crossing an inbred with another,distinct plant such as another inbred. Seed of an F1 hybrid maize plantand an F1 hybrid maize plant and seed thereof are provided.

The genetic complement of the maize plant variety designated PH4D3N isprovided. The phrase “genetic complement” is used to refer to theaggregate of nucleotide sequences, the expression of which sequencesdefines the phenotype of, in the present case, a maize plant, or a cellor tissue of that plant. A genetic complement thus represents thegenetic make-up of an inbred cell, tissue or plant, and a hybrid geneticcomplement represents the genetic make-up of a hybrid cell, tissue orplant. Maize plant cells that have a genetic complement in accordancewith the inbred maize plant cells disclosed herein, and plants, seedsand diploid plants containing such cells are provided.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that variety PH4D3N could be identified by any of themany well-known techniques used for genetic profiling disclosed herein.

In another aspect, hybrid genetic complements are provided, asrepresented by maize plant cells, tissues, plants, and seeds, formed bythe combination of a haploid genetic complement of an inbred maize plantdisclosed herein with a haploid genetic complement of a second maizeplant, such as, another, distinct inbred maize plant. In another aspect,a maize plant regenerated from a tissue culture that comprises a hybridgenetic complement of the inbred maize plant disclosed herein.

Methods of producing an inbred maize plant derived from the maizevariety PH4D3N are provided, the method comprising the steps of: (a)preparing a progeny plant derived from maize variety PH4D3N, whereinsaid preparing comprises crossing a plant of the maize variety PH4D3Nwith a second maize plant; (b) crossing the progeny plant with itself ora second plant to produce a seed of a progeny plant of a subsequentgeneration; (c) repeating steps (a) and (b) with sufficient inbreedinguntil a seed of an inbred maize plant derived from the variety PH4D3N isproduced. In the method, it may be desirable to select particular plantsresulting from step (c) for continued crossing according to steps (b)and (c). By selecting plants having one or more desirable traits, aninbred maize plant derived from the maize variety PH4D3N is obtainedwhich possesses some of the desirable traits of maize variety PH4D3N aswell as potentially other selected traits.

DETAILED DESCRIPTION

A new and distinctive maize inbred variety designated PH4D3N, which hasbeen the result of years of careful breeding and selection in acomprehensive maize breeding program is provided.

Definitions

Maize (Zea mays) can be referred to as maize or corn. Certaindefinitions used in the specification are provided below. In order toprovide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided. NOTE: ABS is in absolute terms and % MN ispercent of the mean for the experiments in which the inbred or hybridwas grown. PCT designates that the trait is calculated as a percentage.% NOT designates the percentage of plants that did not exhibit a trait.For example, STKLDG % NOT is the percentage of plants in a plot thatwere not stalk lodged. These designators will follow the descriptors todenote how the values are to be interpreted.

ABIOTIC STRESS TOLERANCE: resistance to non-biological sources of stressconferred by traits such as nitrogen utilization efficiency, alterednitrogen responsiveness, drought resistance, cold, and salt resistance

ABTSTK=ARTIFICIAL BRITTLE STALK: A count of the number of “snapped”plants per plot following machine snapping. A snapped plant has itsstalk completely snapped at a node between the base of the plant and thenode above the ear. Expressed as percent of plants that did not snap.

ALLELE: Any of one or more alternative forms of a genetic sequence. In adiploid cell or organism, the two alleles of a given sequence typicallyoccupy corresponding loci on a pair of homologous chromosomes.

ANTHESIS: The time of a flower's opening.

ANTIOXIDANT: A chemical compound or substance that inhibits oxidation,including but not limited to tocopherol or tocotrienols.

ANT ROT=ANTHRACNOSE STALK ROT (Colletotrichum graminicola): A 1 to 9visual rating indicating the resistance to Anthracnose Stalk Rot. Ahigher score indicates a higher resistance. Data are collected only whensufficient selection pressure exists.

BACKCROSSING: Process in which a breeder crosses a hybrid progenyvariety back to one of the parental genotypes one or more times.

BACKCROSS PROGENY: Progeny plants produced by crossing one maize line(recurrent parent) with plants of another maize line (donor) thatcomprise a desired trait or locus, selecting progeny plants thatcomprise the desired trait or locus, and crossing them with therecurrent parent one or more times to produce backcross progeny plantsthat comprise said trait or locus.

BARPLT=BARREN PLANTS: The percent of plants per plot that were notbarren (lack ears).

BORBMN=ARTIFICIAL BRITTLE STALK MEAN: The mean percent of plants not“snapped” in a plot following artificial selection pressure. A snappedplant has its stalk completely snapped at a node between the base of theplant and the node above the ear. Expressed as percent of plants thatdid not snap. A higher number indicates better tolerance to brittlesnapping.

BREEDING CROSS: A cross to introduce new genetic material into a plantfor the development of a new variety. For example, one could cross plantA with plant B, wherein plant B would be genetically different fromplant A. After the breeding cross, the resulting F1 plants could then beselfed or sibbed for one, two, three or more times (F1, F2, F3, etc.)until a new inbred variety is developed.

CELL: Cell as used herein includes a plant cell, whether isolated, intissue culture or incorporated in a plant or plant part.

CLDTST=COLD TEST: The percent of plants that germinate under cold testconditions.

CLN=CORN LETHAL NECROSIS: Synergistic interaction of maize chloroticmottle virus (MCMV) in combination with either maize dwarf mosaic virus(MDMV-A or MDMV-B) or wheat streak mosaic virus (WSMV). A 1 to 9 visualrating indicating the resistance to Corn Lethal Necrosis. A higher scoreindicates a higher resistance. Data are collected only when sufficientselection pressure exists.

CMSMT=COMMON SMUT: This is the percentage of plants not infected withCommon Smut. Data are collected only when sufficient selection pressureexists.

COMRST=COMMON RUST (Puccinia sorghi): A 1 to 9 visual rating indicatingthe resistance to Common Rust. A higher score indicates a higherresistance. Data are collected only when sufficient selection pressureexists.

CROSS POLLINATION: Fertilization by the union of two gametes fromdifferent plants.

CROSSING: The combination of genetic material by traditional methodssuch as a breeding cross or backcross, but also including protoplastfusion and other molecular biology methods of combining genetic materialfrom two sources.

D/D=DRYDOWN: This represents the relative rate at which a hybrid willreach acceptable harvest moisture compared to other hybrids on a 1 to 9rating scale. A high score indicates a hybrid that dries relatively fastwhile a low score indicates a hybrid that dries slowly.

DIPERS=DIPLODIA EAR MOLD SCORES (Diplodia maydis and Diplodiamacrospora): A 1 to 9 visual rating indicating the resistance toDiplodia Ear Mold. A higher score indicates a higher resistance. Dataare collected only when sufficient selection pressure exists.

DIPLOID PLANT PART: Refers to a plant part or cell that has a samediploid genotype.

DIPROT=DIPLODIA STALK ROT SCORE: Score of stalk rot severity due toDiplodia (Diplodia maydis). Expressed as a 1 to 9 score with 9 beinghighly resistant. Data are collected only when sufficient selectionpressure exists.

D/T=DROUGHT TOLERANCE: This represents a 1 to 9 rating for droughttolerance, and is based on data obtained under stress conditions. A highscore indicates good drought tolerance and a low score indicates poordrought tolerance. Data are collected only when sufficient selectionpressure exists.

EARMLD=GENERAL EAR MOLD: Visual rating (1 to 9 score) where a 1 is verysusceptible and a 9 is very resistant. This is based on overall ratingfor ear mold of mature ears without determining the specific moldorganism, and may not be predictive for a specific ear mold. Data arecollected only when sufficient selection pressure exists.

EARHT=EDEARHT=EAR HEIGHT: The ear height is a measure from the ground tothe highest placed developed ear node attachment and is measured ininches (EARHT) or cm (EDEARHT).

EARSZ=EAR SIZE: A 1 to 9 visual rating of ear size. The higher therating the larger the ear size.

EDANTCOLs=ANTHER COLOR: Rated on a 1 to 7 scale where 1 is green, 2 isyellow, 3 is pink, 5 is red, and 7 is purple.

EDantants=ANTHER ANTHOCYANIN COLOR INTENSITY: A measure of antheranthocyanin color intensity rated on a 1 to 9 scale where 1 is absent orvery weak, 3 is weak, 5 is medium, 7 is strong, and 9 is very strong.Observed in the middle third of the main branch on fresh anthers.

EDbarants=GLUME ANTHOCYANIN COLORATION AT BASE (WHOLE PLANT, EARINSERTION LEVEL): A measure of the color intensity at the base of theglume, rated on a 1 to 9 scale where 1 is absent or very weak, 3 isweak, 5 is medium, 7 is strong, and 9 is very strong. Observed in themiddle third of the main branch of the tassel.

EDBARCOLs=BAR GLUME COLOR INTENSITY: A measure of the bar glume colorintensity. Bar glume is a dark purple band that may occur on the bottomof a glume. Bar glume color intensity is measured on a scale of 1 to 7where 1 is absent, 2 is weak, 3 is medium, 5 is strong, and 7 is verystrong.

EDBRROANTs=BRACE ROOTS ANTHOCYANIN COLORATION: A measure of the colorintensity of the brace roots rated on a 1 to 9 scale where 1 is absentor very weak, 3 is weak, 5 is medium, 7 is strong, and 9 is very strong.Observed when well developed and fresh brace roots are present on 50% ofplants.

EDCOBAINTs=COB GLUME ANTHOCYANIN COLOR INTENSITY: Rated on a 1 to 9scale where 1 is absent or very weak, 3 is weak, 5 is medium, 7 isstrong, and 9 is very strong. Anthocyanin coloration should be observedon the middle third of the uppermost cob, after the removal of some ofthe grains.

EDCOBCOLs=COB COLOR: A measure of the intensity of pink or salmoncoloration of the cob, rated on a 1 to 9 scale where 1 is absent orwhite, 2 is light pink, 3 is pink, 4 is medium red, 5 is red, 6 ismedium red, 7 is dark red, 8 is dark to very dark red, and 9 is present.

EDCOBDIA=COB DIAMETER: Measured in mm.

EDCOBICAs=COB ANTHOCYANIN COLOR INTENSITY: A measure of the intensity ofpink or salmon coloration of the cob, rated on a 1 to 9 scale where 1 isvery weak, 3 is weak, 5 is medium, 7 is strong, and 9 is very strong.

EDEARDIA=EAR DIAMETER: Measured in mm.

EDEARHULs=EAR HUSK LENGTH: A measure of ear husk length rated on a 1 to9 scale where 1 is very short, 3 is short, 5 is medium, 7 is long, and 9is very long.

EDEARLNG=EAR LENGTH: Measured in mm.

EDEARROW=NUMBER OF ROWS OF GRAIN ON EAR.

EDEARSHAs=EAR SHAPE (TAPER): Rated on a 1 to 3 scale where 1 is conical,2 is conico-cylindrical, and 3 is cylindrical.

EDEARSHLs=EAR SHANK LENGTH SCALE: A measure of the length of the earshank or peduncle, rated on a 1 to 9 scale where 1 is very short, 3 isshort, 5 is medium, 7 is long, 9 is very long.

EDFILEANs=SHEATH ANTHOCYANIN COLOR INTENSITY AT FIRST LEAF STAGE: Ameasure of the anthocyanin color intensity of the sheath of the firstleaf, rated on a 1 to 9 scale where 1 is absent or very weak, 3 is weak,5 is medium, 7 is strong, and 9 is very strong.

EDFILECOs=FOLIAGE INTENSITY OF GREEN COLOR: A measure of the greencoloration intensity in the leaves, rated on a 1 to 3 scale where 1 islight, 2 is medium, and 3 is dark.

EDFILESHs=LEAF TIP SHAPE: An indication of the shape of the apex of thefirst leaf, rated on a 1 to 5 scale where 1 is pointed, 2 is pointed torounded, 3 is rounded, 4 is rounded to spatulate, and 5 is spatulate.

EDGLUANTs=GLUME ANTHOCYANIN COLOR EXCLUDING BASE: A measure of the colorintensity of the glume excluding the base, rated on a 1 to 9 scale where1 is absent or very weak, 3 is weak, 5 is medium, 7 is strong, and 9 isvery strong. Observed in the middle third of the main branch of thetassel.

EDGLUCOLs=GLUME COLOR: Rated on a 1 to 7 scale where 1 is green, 2 isyellow, 3 is pink, 5 is red, and 7 is purple.

EDKERDOCs=DORSAL SIDE OF GRAIN COLOR: Rated on a 1 to 10 scale where 1is white, 2 is yellowish white, 3 is yellow, 4 is yellow orange, 5 isorange, 6 is red orange, 7 is red, 8 is purple, 9 is brownish, and 10 isblue black. Observed in the middle third of the uppermost ear when welldeveloped.

EDKERSHAs=KERNEL SHAPE: Rated on a 1 to 3 scale where 1 is round, 2 iskidney-shaped, and 3 is cuneiform.

EDKERTCOs=TOP OF GRAIN COLOR: Rated on a 1 to 10 scale where 1 is white,2 is yellowish white, 3 is yellow, 4 is yellow orange, 5 is orange, 6 isred orange, 7 is red, 8 is purple, 9 is brownish, and 10 is blue black.Observed in the middle third of the uppermost ear when well developed.

EDLEAANGs=LEAF ANGLE BETWEEN BLADE AND STEM: A measure of the angleformed between stem and leaf, rated on a 1 to 9 scale where 1 is verysmall (<5 degrees), 3 is small (6 to 37 degrees), 5 is medium (38 to 62degrees), 7 is large (63 to 90 degrees), and 9 is very large (>90degrees). Observed on the leaf just above the upper ear.

EDLEAATTs=LEAF ATTITUDE OF ENTIRE PLANT: A measure of leaf curvature orattitude, rated on a 1 to 9 scale where 1 is absent or very slightlyrecurved, 3 is slightly recurved, 5 is moderately recurved, 7 isstrongly recurved, and 9 is very strongly recurved. Observed on the leafjust above the upper ear.

EDLEALNGs=LEAF LENGTH SCORE: A measure of leaf length rated on a 1 to 9scale where 1 indicates <0.70 m, 3 indicates 0.70 m to 0.80 m, 5indicates 0.80 m to 0.90 m, 7 indicates 0.90 m to 1 m, and 9indicates >1.00 m.

EDLEAWID=LEAF WIDTH OF BLADE: A measure of the average leaf width in cm.

EDLELIANTs=LEAF LIMB ANTHOCYANIN COLOR INTENSITY OF ENTIRE PLANT: Ameasure of the leaf limb anthocyanin coloration, rated on a 1 to 9 scalewith 1 being absent or very weak, 3 being weak, 5 being medium, 7 beingstrong, and 9 being very strong.

EDNODANTS=NODES ANTHOCYANIN COLOR INTENSITY: A measure of theanthocyanin coloration of nodes, rated on a 1 to 9 scale where 1 isabsent or very weak, 3 is weak, 5 is medium, 7 is strong, and 9 is verystrong.

EDRATIOEP=RATIO HEIGHT OF INSERTION OF PEDUNCLE OF UPPER EAR TO PLANTLENGTH.

EDSHEAHAs=LEAF SHEATH HAIRNESS SCALE: Rated on a 1 to 6 scale where 1indicates none and 6 indicates fuzzy.

EDSHEAANTs=SHEATH ANTHOCYANIN COLOR INTENSITY: Rated on a 1 to 9 scalewhere 1 is absent or very weak, 3 is weak, 5 is medium, 7 is strong, and9 is very strong.

EDSLKAINTs=SILK ANTHOCYANIN COLOR INTENSITY: A measure of the colorintensity of the silks, rated on a 1 to 9 scale where 1 is absent orvery weak, 3 is weak, 5 is medium, 7 is strong, and 9 is very strong.

EDSTLANTs=INTERNODE ANTHOCYANIN COLOR INTENSITY: A measure ofanthocyanin coloration of nodes, rated on a 1 to 9 scale where 1 isabsent or very weak, 3 is weak, 5 is medium, 7 is strong, and 9 is verystrong. Observed just above the insertion point of the peduncle of theupper ear.

EDTA1RYATs=TASSEL LATERAL BRANCH CURVATURE: Rated on a 1 to 9 scalewhere 1 indicates absent or very slightly recurved (<5 degrees), 3indicates slightly recurved (6 to 37 degrees), 5 indicates moderatelyrecurved (38 to 62 degrees), 7 indicates strongly recurved (63 to 90degrees), and 9 indicates very strongly recurved (>90 degrees). Observedon the second branch from the bottom of the tassel.

EDTA1RYBRs=NUMBER OF PRIMARY LATERAL TASSEL BRANCHES: Rated on a 1 to 9scale where 1 indicates absent or very few (<4 branches), 3 indicatesfew (4 to 10), 5 indicates medium (11 to 15), 7 indicates many (16 to20), and 9 indicates very many (>20).

EDTASAHB=LENGTH OF MAIN AXIS ABOVE HIGHEST LATERAL BRANCH: Length of thetassel's main axis above the highest lateral branch in cm.

EDTASANGs=TASSEL ANGLE BETWEEN MAIN AXIS AND LATERAL BRANCHES: Rated ona 1 to 9 scale where 1 is very small (<5 degrees), 3 is small (6 to 37degrees), 5 is medium (38 to 62 degrees), 7 is large (63 to 90 degrees),and 9 is very large (>90 degrees). Observed on second branch from bottomof tassel.

EDTASEBRs=SECONDARY TASSEL BRANCHES (NUMBER): The number of secondarytassel branches, rated on a 1 to 7 scale where 1 indicates 0 to 3branches, 2 indicates 4 to 10, 3 indicates 11 to 15, 5 indicates 16 to20, and 7 indicates >20.

EDTASLPBRs=PRIMARY TASSEL BRANCH LENGTH: A measure of the length of theprimary or lateral tassel branch, rated on a 1 to 9 scale where 1 isvery short, 3 is short, 5 is medium, 7 is long, 9 is very long. Observedon the second branch from the bottom of the tassel.

EDTASULB=LENGTH OF MAIN AXIS ABOVE LOWEST LATERAL BRANCH: The length ofthe tassel's main axis above the lowest lateral branch in cm.

EDZIGZAGs=DEGREE OF STEM ZIG-ZAG: Rated on a scale of 1 to 3 where 1 isabsent or very slight, 2 is slight, and 3 is strong.

EGRWTH=EARLY GROWTH: This is a measure of the relative height and sizeof a corn seedling at the 2-4 leaf stage of growth. This is a visualrating (1 to 9), with 1 being weak or slow growth, 5 being averagegrowth and 9 being strong growth. Taller plants, wider leaves, moregreen mass and darker color constitute higher score. Data are collectedonly when sufficient selection pressure exists.

ERTLPN=EARLY ROOT LODGING: An estimate of the percentage of plants thatdo not root lodge prior to or around anthesis; plants that lean from thevertical axis at an approximately 30° angle or greater would beconsidered as root lodged. Data are collected only when sufficientselection pressure exists.

ERTLSC=EARLY ROOT LODGING SCORE: Score for severity of plants that leanfrom a vertical axis at an approximate 30° angle or greater whichtypically results from strong winds prior to or around floweringrecorded within 2 weeks of a wind event. Expressed as a 1 to 9 scorewith 9 being no lodging. Data are collected only when sufficientselection pressure exists.

ESTCNT=EARLY STAND COUNT: This is a measure of the stand establishmentin the spring and represents the number of plants that emerge on perplot basis for the inbred or hybrid.

EYESPT=EYE SPOT (Kabatiella zeae or Aureobasidium zeae): A 1 to 9 visualrating indicating the resistance to Eye Spot. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists.

F1 PROGENY: A progeny plant produced by crossing a plant of one maizeline with a plant of another maize line.

FUSERS=FUSARIUM EAR ROT SCORE (Fusarium moniliforme or Fusariumsubglutinans): A 1 to 9 visual rating indicating the resistance toFusarium Ear Rot. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists.

GDU=GROWING DEGREE UNITS: Using the Barger Heat Unit Theory, whichassumes that maize growth occurs in the temperature range 50° F.-86° F.and that temperatures outside this range slow down growth; the maximumdaily heat unit accumulation is 36 and the minimum daily heat unitaccumulation is 0. The seasonal accumulation of GDU is a major factor indetermining maturity zones.

GDUSHD=EDDAYSH=GDU TO SHED: The number of growing degree units (GDUs) orheat units required for an inbred variety or hybrid to haveapproximately 50 percent of the plants shedding pollen and is measuredfrom the time of planting. Growing degree units are calculated by theBarger Method, where the heat units for a 24-hour period are:

${GDU} = {\frac{\left( {{Max}.{temp}.{+ {{Min}.{temp}.}}} \right)}{2} - 50}$

The units determined by the Barger Method are then divided by 10. Thehighest maximum temperature used is 86° F. and the lowest minimumtemperature used is 50° F. For each inbred or hybrid it takes a certainnumber of GDUs to reach various stages of plant development.

GDUSLK=EDDAYSLK=GDU TO SILK: The number of growing degree units requiredfor an inbred variety or hybrid to have approximately 50 percent of theplants with silk emergence from time of planting. Growing degree unitsare calculated by the Barger Method as given in GDUSHD definition andthen divided by 10.

GIBERS=GIBBERELLA EAR ROT (PINK MOLD) (Gibberella zeae): A 1 to 9 visualrating indicating the resistance to Gibberella Ear Rot. A higher scoreindicates a higher resistance. Data are collected only when sufficientselection pressure exists.

GIBROT=GIBBERELLA STALK ROT SCORE: Score of stalk rot severity due toGibberella (Gibberella zeae). Expressed as a 1 to 9 score with 9 beinghighly resistant. Data are collected only when sufficient selectionpressure exists.

GLFSPT=GRAY LEAF SPOT (Cercospora zeae-maydis): A 1 to 9 visual ratingindicating the resistance to Gray Leaf Spot. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists.

GOSWLT=GOSS' WILT (Corynebacterium nebraskense): A 1 to 9 visual ratingindicating the resistance to Goss' Wilt. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists.

GRAIN TEXTURE: A visual rating used to indicate the appearance of maturegrain observed in the middle third of the uppermost ear when welldeveloped. Grain or seed with a hard grain texture is indicated asflint; grain or seed with a soft grain texture is indicted as dent.Medium grain or seed texture may be indicated as flint-dent orintermediate. Other grain textures include flint-like, dent-like, sweet,pop, waxy and flour.

GRNAPP=GRAIN APPEARANCE: This is a 1 to 9 rating for the generalappearance of the shelled grain as it is harvested based on such factorsas the color of harvested grain, any mold on the grain, and any crackedgrain. Higher scores indicate better grain visual quality.

HAPLOID PLANT PART: A plant part or cell having a haploid genotype.

HCBLT=HELMINTHOSPORIUM CARBONUM LEAF BLIGHT (Helminthosporium carbonum):A 1 to 9 visual rating indicating the resistance to Helminthosporiuminfection. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists.

HD SMT=HEAD SMUT (Sphacelotheca reiliana): This indicates the percentageof plants not infected. Data are collected only when sufficientselection pressure exists.

HSKCVR=HUSK COVER: A 1 to 9 score based on performance relative to keychecks, with a score of 1 indicating very short husks, tip of ear andkernels showing; 5 is intermediate coverage of the ear under mostconditions, sometimes with thin husk; and a 9 has husks extending andclosed beyond the tip of the ear. Scoring can best be done nearphysiological maturity stage or any time during dry down untilharvested.

HYBRID VARIETY: A substantially heterozygous hybrid line and minorgenetic modifications thereof that retain the overall genetics of thehybrid line.

INBRED: A variety developed through inbreeding or doubled haploidy thatpreferably comprises homozygous alleles at about 95% or more of itsloci. An inbred can be reproduced by selfing or growing in isolation sothat the plants can only pollinate with the same inbred variety.

INTROGRESSION: The process of transferring genetic material from onegenotype to another.

KERNEL PERICARP COLOR is scored when kernels have dried down and istaken at or about 65 days after 50% silk. Score codes are: Colorless=1;Red with white crown=2; Tan=3; Bronze=4; Brown=5; Light red=6; Cherryred=7.

KER_WT=KERNEL NUMBER PER UNIT WEIGHT (Pounds or Kilograms): The numberof kernels in a specific measured weight; determined after removal ofextremely small and large kernels.

LINKAGE: Refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent.

LINKAGE DISEQUILIBRIUM: Refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

LOCUS: A specific location on a chromosome.

LOCUS CONVERSION (Also called TRAIT CONVERSION): A locus conversionrefers to plants within a variety that have been modified in a mannerthat retains the overall genetics of the variety and further comprisesone or more loci with a specific desired trait, such as male sterility,insect resistance, disease resistance or herbicide tolerance orresistance. Examples of single locus conversions include mutant genes,transgenes and native traits finely mapped to a single locus. One ormore locus conversion traits may be introduced into a single cornvariety.

L/POP=YIELD AT LOW DENSITY: Yield ability at relatively low plantdensities on a 1 to 9 relative system with a higher number indicatingthe hybrid responds well to low plant densities for yield relative toother hybrids. A 1, 5, and 9 would represent very poor, average, andvery good yield response, respectively, to low plant density.

LRTLPN=LATE ROOT LODGING: An estimate of the percentage of plants thatdo not root lodge after anthesis through harvest; plants that lean fromthe vertical axis at an approximately 30° angle or greater would beconsidered as root lodged. Data are collected only when sufficientselection pressure exists.

LRTLSC=LATE ROOT LODGING SCORE: Score for severity of plants that leanfrom a vertical axis at an approximate 30° angle or greater whichtypically results from strong winds after flowering. Recorded prior toharvest when a root-lodging event has occurred. This lodging results inplants that are leaned or “lodged” over at the base of the plant and donot straighten or “goose-neck” back to a vertical position. Expressed asa 1 to 9 score with 9 being no lodging. Data are collected only whensufficient selection pressure exists.

MALE STERILITY: A male sterile plant is one which produces no viablepollen no (pollen that is able to fertilize the egg to produce a viableseed). Male sterility prevents self pollination. These male sterileplants are therefore useful in hybrid plant production.

MDMCPX=MAIZE DWARF MOSAIC COMPLEX (MDMV=Maize Dwarf Mosaic Virus andMCDV=Maize Chlorotic Dwarf Virus): A 1 to 9 visual rating indicating theresistance to Maize Dwarf Mosaic Complex. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists.

MILKLN=percent milk in mature grain.

MST=HARVEST MOISTURE: The moisture is the actual percentage moisture ofthe grain at harvest.

NEI DISTANCE: A quantitative measure of percent similarity between twovarieties. Nei's distance between varieties A and B can be defined as1−(2*number alleles in common/(number alleles in A+number alleles in B).For example, if varieties A and B are the same for 95 out of 100alleles, the Nei distance would be 0.05. If varieties A and B are thesame for 98 out of 100 alleles, the Nei distance would be 0.02. Freesoftware for calculating Nei distance is available on the internet atmultiple locations. See Nei, Proc Natl Acad Sci, 76:5269-5273 (1979.

NLFBLT=NORTHERN LEAF BLIGHT (Helminthosporium turcicum or Exserohilumturcicum): A 1 to 9 visual rating indicating the resistance to NorthernLeaf Blight. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists.

OILT=GRAIN OIL: Absolute value of oil content of the kernel as predictedby Near-Infrared Transmittance and expressed as a percent of dry matter.

PERCENT IDENTITY: Percent identity as used herein refers to thecomparison of the alleles present in two varieties. For example, whencomparing two inbred plants to each other, each inbred plant will havethe same allele (and therefore be homozygous) at almost all of theirloci. Percent identity is determined by comparing a statisticallysignificant number of the homozygous alleles of two varieties. Forexample, a percent identity of 90% between PH4D3N and other varietymeans that the two varieties have the same homozygous alleles at 90% oftheir loci.

PLANT: As used herein, the term “plant” includes reference to animmature or mature whole plant, including a plant that has beendetasseled or from which seed or grain has been removed. Seed or embryothat will produce the plant is also considered to be the plant.

PLANT PART: As used herein, the term “plant part” includes leaves,stems, roots, seed, grain, embryo, pollen, ovules, flowers, ears, cobs,husks, stalks, root tips, anthers, pericarp, silk, tissue, cells and thelike. In some embodiments, the plant part contains at least one cell ofinbred maize variety PH4D3N (or a locus conversion thereof) or a hybridproduced from inbred variety PH4D3N (or a locus conversion thereof).

PLATFORM indicates the variety with the base genetics and the varietywith the base genetics comprising locus conversion(s). There can be aplatform for the inbred maize variety and the hybrid maize variety.

PLTHT=EDPLTHWT=PLANT HEIGHT: This is a measure of the height of theplant from the ground to the tip of the tassel in inches (PLTHT) or cm.

POLSC=POLLEN SCORE: A 0 to 9 visual rating indicating the amount ofpollen shed. The higher the score the more pollen shed.

RM=RELATIVE MATURITY: This is a predicted relative maturity, based onthe harvest moisture of the grain. The relative maturity rating is basedon a known set of checks and utilizes standard linear regressionanalyses and is also referred to as the Comparative Relative MaturityRating System that is similar to the Minnesota Relative Maturity RatingSystem.

PROT=GRAIN PROTEIN: Absolute value of protein content of the kernel aspredicted by Near-Infrared Transmittance and expressed as a percent ofdry matter.

RESISTANCE: Synonymous with tolerance. The ability of a plant towithstand exposure to an insect, disease, herbicide or other condition.A resistant plant variety will have a level of resistance higher than acomparable wild-type variety.

RTLDG=ROOT LODGING: Root lodging is the percentage of plants that do notroot lodge; plants that lean from the vertical axis at an approximately30° angle or greater would be counted as root lodged. Data are collectedonly when sufficient selection pressure exists.

SEED: Fertilized and ripened ovule, consisting of the plant embryo,stored food material, and a protective outer seed coat. Synonymous withgrain.

SELF POLLINATION: A plant is self-pollinated if pollen from one floweris transferred to the same or another flower of the same plant.

SIB POLLINATION: A plant is sib-pollinated when individuals within thesame family or variety are used for pollination.

SITE SPECIFIC INTEGRATION: Genes that create a site for site specificDNA integration. This includes the introduction of FRT sites that may beused in the FLP/FRT system and/or Lox sites that may be used in theCre/Loxp system. For example, see Lyznik, et al., Site-SpecificRecombination for Genetic Engineering in Plants, Plant Cell Rep (2003)21:925-932 and WO 99/25821.

SLFBLT=SOUTHERN LEAF BLIGHT (Helminthosporium maydis or Bipolarismaydis): A 1 to 9 visual rating indicating the resistance to SouthernLeaf Blight. A higher score indicates a higher resistance. Data arecollected only when sufficient selection pressure exists.

SNP=SINGLE-NUCLEOTIDE POLYMORPHISM: is a DNA sequence variationoccurring when a single nucleotide in the genome differs betweenindividual plant or plant varieties. The differences can be equated withdifferent alleles, and indicate polymorphisms. A number of SNP markerscan be used to determine a molecular profile of an individual plant orplant variety and can be used to compare similarities and differencesamong plants and plant varieties.

SOURST=SOUTHERN RUST (Puccinia polysora): A 1 to 9 visual ratingindicating the resistance to Southern Rust. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists.

SPKDSC=EDTASAFDs=TASSEL SPIKELET DENSITY SCORE: The visual rating of howdense spikelets are on the middle to middle third of tassel branches. Ahigher score on a 1-9 scale indicates higher spikelet density (SPKDSC).On a 3 to 7 scale, 3 is moderately lax, 5 is medium, and 7 is moderatelydense.

STAGRN=STAY GREEN: Stay green is the measure of plant health near thetime of black layer formation (physiological maturity). A high scoreindicates better late-season plant health.

STKLDS=STALK LODGING SCORE: A plant is considered as stalk lodged if thestalk is broken or crimped between the ear and the ground. This can becaused by any or a combination of the following: strong winds late inthe season, disease pressure within the stalks, ECB damage orgenetically weak stalks. This trait should be taken just prior to or atharvest. Expressed on a 1 to 9 scale with 9 being no lodging. Data arecollected only when sufficient selection pressure exists.

STLLPN=LATE STALK LODGING: This is the percent of plants that did notstalk lodge (stalk breakage or crimping) at or around late seasonharvest (when grain moisture is below 20%) as measured by either naturallodging or pushing the stalks and determining the percentage of plantsthat break or crimp below the ear. Data are collected only whensufficient selection pressure exists.

STLPCN=STALK LODGING REGULAR: This is an estimate of the percentage ofplants that did not stalk lodge (stalk breakage) at regular harvest(when grain moisture is between about 20% and 30%) as measured by eithernatural lodging or pushing the stalks and determining the percentage ofplants that break below the ear. Data are collected only when sufficientselection pressure exists.

STWWLT=Stewart's Wilt (Erwinia stewartii): A 1 to 9 visual ratingindicating the resistance to Stewart's Wilt. A higher score indicates ahigher resistance. Data are collected only when sufficient selectionpressure exists.

SSRs: Genetic markers based on polymorphisms in repeated nucleotidesequences, such as microsatellites. A marker system based on SSRs can beinformative in linkage analysis relative to other marker systems in thatmultiple alleles may be present.

TASBRN=TASSEL BRANCH NUMBER: The number of tassel branches, with anthersoriginating from the central spike.

TASSZ=TASSEL SIZE: A 1 to 9 visual rating was used to indicate therelative size of the tassel. A higher rating means a larger tassel.

TAS WT=TASSEL WEIGHT: This is the average weight of a tassel (grams)just prior to pollen shed.

TILLER=TILLERS: A count of the number of tillers per plot that couldpossibly shed pollen was taken. Data are given as a percentage oftillers: number of tillers per plot divided by number of plants perplot. A tiller is defined as a secondary shoot that has developed as atassel capable of shedding pollen.

TSTWT=TEST WEIGHT (ADJUSTED): The measure of the weight of grain inpounds for a given volume (bushel), adjusted for MST less than or equalto 22%.

TSTWTN=TEST WEIGHT (UNADJUSTED): The measure of the weight of the grainin pounds for a given volume (bushel).

VARIETY: A maize line and minor genetic modifications thereof thatretain the overall genetics of the line including but not limited to alocus conversion, a mutation, or a somoclonal variant.

YIELD BU/A=YIELD (BUSHELS/ACRE): Yield of the grain at harvest by weightor volume (bushels) per unit area (acre) adjusted to 15% moisture.

YLDADV=YIELD ADVANTAGE: The yield advantage of variety #1 over variety#2 as calculated by: YIELD of variety #1−YIELD variety #2=YIELDADVANTAGE of variety #1.

YIELDMST=YIELD/MOISTURE RATIO.

YLDSC=YIELD SCORE: A 1 to 9 visual rating was used to give a relativerating for yield based on plot ear piles. The higher the rating thegreater visual yield appearance.

YIELDS=Silage Dry Matter Yield (tons/acre @ 100% DM)

MLKYLD=Estimated pounds of milk produced per ton of dry matter fed andis based on utilizing nutrient content and fiber digestibility

ADJMLK=Estimated pounds of milk produced per acre of silage dry matterbased on an equation and is MLKYLD divided by YIELDS.

SLGPRM=Silage Predicted Relative Maturity

SY30DM=Silage Yield (Tonnage) Adjusted to 30% Dry Matter

PCTMST=Silage Harvest Moisture %

NDFDR=Silage Fiber Digestibility Based on rumen fluid NIRS calibration

NDFDC=Silage Fiber Digestibility Based on rumen fluid NIRS calibration

Phenotypic Characteristics of PH4D3N

Inbred maize variety PH4D3N may be used as a male or female in theproduction of the first generation F1 hybrid. The variety has shownuniformity and stability within the limits of environmental influencefor all the traits as described in the Variety Description Information(Table 1, found at the end of the section). The variety has beenself-pollinated and ear-rowed a sufficient number of generations withcareful attention paid to uniformity of plant type to ensure sufficienthomozygosity and phenotypic stability for use in commercial hybrid seedproduction. The variety has been increased both by hand and in isolatedfields with continued observation for uniformity. No variant traits havebeen observed or are expected in PH4D3N.

Genotypic Characteristics of PH4D3N

In addition to phenotypic observations, a plant can also be identifiedby its genotype. The genotype of a plant can be characterized through agenetic marker profile.

As a result of inbreeding, PH4D3N is substantially homozygous. Thishomozygosity can be characterized at the loci shown in a marker profile.An F1 hybrid made with PH4D3N would substantially comprise the markerprofile of PH4D3N. This is because an F1 hybrid is the sum of its inbredparents, e.g., if one inbred parent is homozygous for allele x at aparticular locus, and the other inbred parent is homozygous for allele yat that locus, the F1 hybrid will be xy (heterozygous) at that locus. Agenetic marker profile can therefore be used to identify hybridscomprising PH4D3N as a parent, since such hybrids will comprise two setsof alleles, one set of which will be from PH4D3N. The determination ofthe male set of alleles and the female set of alleles may be made byprofiling the hybrid and the pericarp of the hybrid seed, which iscomposed of maternal parent cells. One way to obtain the paternal parentprofile is to subtract the pericarp profile from the hybrid profile.

Subsequent generations of progeny produced by selection and breeding areexpected to be of genotype xx (homozygous), yy (homozygous), or xy(heterozygous) for these locus positions. When the F1 plant is used toproduce an inbred, the resulting inbred should be either x or y for thatallele.

Therefore, in accordance with the above, an embodiment is a PH4D3Nprogeny maize plant or plant part that is a first generation (F1) hybridmaize plant comprising two sets of alleles, wherein one set of thealleles is the same as PH4D3N at substantially all loci. A maize cellwherein one set of the alleles is the same as PH4D3N at substantiallyall loci is also provided. This maize cell may be a part of a hybridseed, plant or plant part produced by crossing PH4D3N with another maizeplant.

Genetic marker profiles can be obtained by techniques such asRestriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), Simple Sequence Repeats (SSRs) which are also referred to asMicrosatellites, and Single Nucleotide Polymorphisms (SNPs). Forexample, see Berry et al. (2002), “Assessing Probability of AncestryUsing Simple Sequence Repeat Profiles: Applications to Maize Hybrids andInbreds”, Genetics, 2002, 161:813-824, and Berry et al. (2003),“Assessing Probability of Ancestry Using Simple Sequence RepeatProfiles: Applications to Maize Inbred Lines and Soybean Varieties”,Genetics, 2003, 165: 331-342.

Particular markers used for these purposes may include any type ofmarker and marker profile which provides a means of distinguishingvarieties. A genetic marker profile can be used, for example, toidentify plants of the same variety or related varieties or to determineor validate a pedigree. In addition to being used for identification ofmaize variety PH4D3N, a hybrid produced through the use of PH4D3N, andthe identification or verification of pedigree for progeny plantsproduced through the use of PH4D3N, a genetic marker profile is alsouseful in developing a locus conversion of PH4D3N.

Methods of isolating nucleic acids from maize plants and methods forperforming genetic marker profiles using SNP and SSR polymorphisms arewell known in the art. SNPs are genetic markers based on a polymorphismin a single nucleotide. A marker system based on SNPs can be highlyinformative in linkage analysis relative to other marker systems in thatmultiple alleles may be present.

A method comprising isolating nucleic acids, such as DNA, from a plant,a plant part, plant cell or a seed of the maize plants disclosed hereinis provided. The method can include mechanical, electrical and/orchemical disruption of the plant, plant part, plant cell or seed,contacting the disrupted plant, plant part, plant cell or seed with abuffer or solvent, to produce a solution or suspension comprisingnucleic acids, optionally contacting the nucleic acids with aprecipitating agent to precipitate the nucleic acids, optionallyextracting the nucleic acids, and optionally separating the nucleicacids such as by centrifugation or by binding to beads or a column, withsubsequent elution, or a combination thereof. If DNA is being isolated,an RNase can be included in one or more of the method steps. The nucleicacids isolated can comprise all or substantially all of the genomic DNAsequence, all or substantially all of the chromosomal DNA sequence orall or substantially all of the coding sequences (cDNA) of the plant,plant part, or plant cell from which they were isolated. The amount andtype of nucleic acids isolated may be sufficient to permit whole genomesequencing of the plant from which they were isolated or chromosomalmarker analysis of the plant from which they were isolated.

The methods can be used to produce nucleic acids from the plant, plantpart, seed or cell, which nucleic acids can be, for example, analyzed toproduce data. The data can be recorded. The nucleic acids from thedisrupted cell, the disrupted plant, plant part, plant cell or seed orthe nucleic acids following isolation or separation can be contactedwith primers and nucleotide bases, and/or a polymerase to facilitate PCRsequencing or marker analysis of the nucleic acids. In some examples,the nucleic acids produced can be sequenced or contacted with markers toproduce a genetic profile, a molecular profile, a marker profile, ahaplotype, or any combination thereof. In some examples, the geneticprofile or nucleotide sequence is recorded on a computer readablemedium. In other examples, the methods may further comprise using thenucleic acids produced from plants, plant parts, plant cells or seeds ina plant breeding program, for example in making crosses, selectionand/or advancement decisions in a breeding program. Crossing includesany type of plant breeding crossing method, including but not limited tocrosses to produce hybrids, outcrossing, selfing, backcrossing, locusconversion, introgression and the like.

Favorable genotypes and or marker profiles, optionally associated with atrait of interest, may be identified by one or more methodologies. Insome examples one or more markers are used, including but not limited toAFLPs, RFLPs, ASH, SSRs, SNPs, indels, padlock probes, molecularinversion probes, microarrays, sequencing, and the like. In somemethods, a target nucleic acid is amplified prior to hybridization witha probe. In other cases, the target nucleic acid is not amplified priorto hybridization, such as methods using molecular inversion probes (see,for example Hardenbol et al. (2003) Nat Biotech 21:673-678). In someexamples, the genotype related to a specific trait is monitored, whilein other examples, a genome-wide evaluation including but not limited toone or more of marker panels, library screens, association studies,microarrays, gene chips, expression studies, or sequencing such aswhole-genome resequencing and genotyping-by-sequencing (GBS) may beused. In some examples, no target-specific probe is needed, for exampleby using sequencing technologies, including but not limited tonext-generation sequencing methods (see, for example, Metzker (2010) NatRev Genet 11:31-46; and, Egan et al. (2012) Am J Bot 99:175-185) such assequencing by synthesis (e.g., Roche 454 pyrosequencing, IIlumina GenomeAnalyzer, and Ion Torrent PGM or Proton systems), sequencing by ligation(e.g., SOLiD from Applied Biosystems, and Polnator system from AzcoBiotech), and single molecule sequencing (SMS or third-generationsequencing) which eliminate template amplification (e.g., Helicossystem, and PacBio RS system from Pacific BioSciences). Furthertechnologies include optical sequencing systems (e.g., Starlight fromLife Technologies), and nanopore sequencing (e.g., GridION from OxfordNanopore Technologies). Each of these may be coupled with one or moreenrichment strategies for organellar or nuclear genomes in order toreduce the complexity of the genome under investigation via PCR,hybridization, restriction enzyme (see, e.g., Elshire et al. (2011) PLoSONE 6:e19379), and expression methods. In some examples, no referencegenome sequence is needed in order to complete the analysis. PH4D3N andits plant parts can be identified through a molecular marker profile.Such plant parts may be either diploid or haploid. The plant partincludes at least one cell of the plant from which it was obtained, suchas a diploid cell, a haploid cell or a somatic cell. Also provided areplants and plant parts substantially benefiting from the use of varietyPH4D3N in their development, such as variety PH4D3N comprising a locusconversion.

Comparing PH4D3N to Other Inbreds

A breeder uses various methods to help determine which plants should beselected from segregating populations and ultimately which inbredvarieties will be used to develop hybrids for commercialization. Inaddition to knowledge of the germplasm and plant genetics, a part of theselection process is dependent on experimental design coupled with theuse of statistical analysis. Experimental design and statisticalanalysis are used to help determine which plants, which family ofplants, and finally which inbred varieties and hybrid combinations aresignificantly better or different for one or more traits of interest.Experimental design methods are used to assess error so that differencesbetween two inbred varieties or two hybrid varieties can be moreaccurately evaluated. Statistical analysis includes the calculation ofmean values, determination of the statistical significance of thesources of variation, and the calculation of the appropriate variancecomponents. Either a five or a one percent significance level iscustomarily used to determine whether a difference that occurs for agiven trait is real or due to the environment or experimental error. Oneof ordinary skill in the art of plant breeding would know how toevaluate the traits of two plant varieties to determine if there is asignificant difference between the two traits expressed by thosevarieties. For example, see Fehr, Walt, Principles of CultivarDevelopment, p. 261-286 (1987). Mean trait values may be used todetermine whether trait differences are significant. Trait values shouldpreferably be measured on plants grown under the same environmentalconditions, and environmental conditions should be appropriate for thetraits or traits being evaluated. Sufficient selection pressure shouldbe present for optimum measurement of traits of interest such asherbicide tolerance, insect or disease resistance. A locus conversion ofPH4D3N for herbicide tolerance should be compared with an isogeniccounterpart in the absence of the converted trait. In addition, a locusconversion for insect or disease resistance should be compared to theisogenic counterpart, in the absence of disease pressure or insectpressure.

Development of Maize Hybrids Using PH4D3N

A single cross maize hybrid results from the cross of two inbredvarieties, each of which has a genotype that complements the genotype ofthe other. A hybrid progeny of the first generation is designated F1. Inthe development of commercial hybrids in a maize plant breeding program,only the F1 hybrid plants are sought. F1 hybrids are more vigorous thantheir inbred parents. This hybrid vigor, or heterosis, can be manifestedin many polygenic traits, including increased vegetative growth andincreased yield.

PH4D3N may be used to produce hybrid maize. One such embodiment is themethod of crossing maize variety PH4D3N with another maize plant, suchas a different maize variety, to form a first generation F1 hybrid seed.The first generation F1 hybrid seed, plant and plant part produced bythis method are provided. The first generation F1 seed, plant and plantpart will comprise an essentially complete set of the alleles of varietyPH4D3N. One of ordinary skill in the art can utilize molecular methodsto identify a particular F1 hybrid plant produced using variety PH4D3N.Further, one of ordinary skill in the art may also produce F1 hybridswith transgenic, male sterile and/or locus conversions of varietyPH4D3N.

The development of a maize hybrid in a maize plant breeding programinvolves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of theselected plants from the breeding crosses for several generations toproduce a series of varieties, such as PH4D3N, which, although differentfrom each other, breed true and are highly uniform; and (3) crossing theselected varieties with different varieties to produce the hybrids.During the inbreeding process in maize, the vigor of the varietiesdecreases, and so one would not be likely to use PH4D3N directly toproduce grain. However, vigor is restored when PH4D3N is crossed to adifferent inbred variety to produce a commercial F1 hybrid. Aconsequence of the homozygosity and homogeneity of the inbred variety isthat the hybrid between a defined pair of inbreds may be reproducedindefinitely as long as the homogeneity of the inbred parents ismaintained.

PH4D3N may be used to produce a single cross hybrid, a double crosshybrid, or a three-way hybrid. A single cross hybrid is produced whentwo inbred varieties are crossed to produce the F1 progeny. A doublecross hybrid is produced from four inbred varieties crossed in pairs(A×B and C×D) and then the two F1 hybrids are crossed again (A×B)×(C×D).A three-way cross hybrid is produced from three inbred varieties wheretwo of the inbred varieties are crossed (A×B) and then the resulting F1hybrid is crossed with the third inbred (A×B)×C. In each case, pericarptissue from the female parent will be a part of and protect the hybridseed.

Molecular data from PH4D3N may be used in a plant breeding process.Nucleic acids may be isolated from a seed of PH4D3N or from a plant,plant part, or cell produced by growing a seed of PH4D3N, or from a seedof PH4D3N with a locus conversion, or from a plant, plant part, or cellof PH4D3N with a locus conversion. One or more polymorphisms may beisolated from the nucleic acids. A plant having one or more of theidentified polymorphisms may be selected and used in a plant breedingmethod to produce another plant.

Combining Ability of PH4D3N

Combining ability of a variety, as well as the performance of thevariety per se, is a factor in the selection of improved maize inbreds.Combining ability refers to a variety's contribution as a parent whencrossed with other varieties to form hybrids. The hybrids formed for thepurpose of selecting superior varieties may be referred to as testcrosses, and include comparisons to other hybrid varieties grown in thesame environment (same cross, location and time of planting). One way ofmeasuring combining ability is by using values based in part on theoverall mean of a number of test crosses weighted by number ofexperiment and location combinations in which the hybrid combinationsoccurs. The mean may be adjusted to remove environmental effects andknown genetic relationships among the varieties.

General combining ability provides an overall score for the inbred overa large number of test crosses. Specific combining ability providesinformation on hybrid combinations formed by PH4D3N and a specificinbred parent. A variety such as PH4D3N which exhibits good generalcombining ability may be used in a large number of hybrid combinations.

Hybrid comparisons represent specific hybrid crosses with PH4D3N and acomparison of these specific hybrids with other hybrids with favorablecharacteristics. These comparisons illustrate the good specificcombining ability of PH4D3N. A specific hybrid for which PH4D3N is aparent is compared with other hybrids. Numerous species of the genus ofF1 hybrids created with PH4D3N have been reduced to practice. Thesecomparisons illustrate the good specific combining ability of PH4D3N orPH4D3N comprising locus conversions.

Introduction of a New Trait or Locus into PH4D3N

Inbred PH4D3N represents a new base genetic variety into which a newlocus or trait may be introduced or introgressed. Transformation andbackcrossing represent two methods that can be used to accomplish suchan introgression. The term locus conversion is used to designate theproduct of such an introgression.

A backcross or locus conversion of PH4D3N occurs when DNA sequences areintroduced through backcrossing (Hallauer et al. in Corn and CornImprovement, Sprague and Dudley, Third Ed. 1998), with PH4D3N utilizedas the recurrent parent. Both naturally occurring, modified andtransgenic DNA sequences may be introduced through backcrossingtechniques. A backcross or locus conversion may produce a plant with atrait or locus conversion in at least one or more backcrosses, includingat least 2 backcrosses, at least 3 backcrosses, at least 4 backcrosses,at least 5 backcrosses and the like. Molecular marker assisted breedingor selection may be utilized to reduce the number of backcrossesnecessary to achieve the backcross conversion. For example, see Openshawet al., “Marker-assisted Selection in Backcross Breeding,” in:Proceedings Symposium of the Analysis of Molecular Data, August 1994,Crop Science Society of America, Corvallis, Oreg., which demonstratedthat a backcross locus conversion can be made in as few as twobackcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (a single gene or closely linked genes comparedto unlinked genes), the level of expression of the trait, the type ofinheritance (cytoplasmic or nuclear), dominant or recessive traitexpression, and the types of parents included in the cross. It isunderstood by those of ordinary skill in the art that for single locusor gene traits that are relatively easy to classify, the backcrossmethod is effective and relatively easy to manage. (See Hallauer et al.in Corn and Corn Improvement, Sprague and Dudley, Third Ed. 1998).Desired traits that may be transferred through backcross conversioninclude, but are not limited to, waxy starch, sterility (nuclear andcytoplasmic), fertility restoration, grain color (white), nutritionalenhancements, drought tolerance, nitrogen utilization, altered fattyacid profile, increased digestibility, low phytate, industrialenhancements, disease resistance (bacterial, fungal, or viral), insectresistance, and herbicide tolerance or resistance. A locus conversion,also called a trait conversion, can be a native trait or a transgenictrait. In addition, an recombination site itself, such as an FRT site,Lox site, or other site specific integration site may be inserted bybackcrossing and utilized for direct insertion of one or more genes ofinterest into a specific plant variety. A single locus may containseveral transgenes, such as a transgene for disease resistance that, inthe same expression vector, also contains a transgene for herbicidetolerance or resistance. The gene for herbicide tolerance or resistancemay be used as a selectable marker and/or as a phenotypic trait. Asingle locus conversion of a site specific integration system allows forthe integration of multiple genes at a known recombination site in thegenome. At least one, at least two or at least three and less than ten,less than nine, less than eight, less than seven, less than six, lessthan five or less than four locus conversions may be introduced into theplant by backcrossing, introgression or transformation to express thedesired trait, while the plant, or a plant grown from the seed, plantpart or plant cell, otherwise retains the phenotypic characteristics ofthe deposited seed when grown under the same environmental conditions.

The backcross or locus conversion may result from either the transfer ofa dominant allele or a recessive allele. Selection of progeny containingthe trait of interest can be accomplished by direct selection for atrait associated with a dominant allele. Transgenes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele, such as the waxy starchcharacteristic, requires growing and selfing the first backcrossgeneration to determine which plants carry the recessive alleles.Recessive traits may require additional progeny testing in successivebackcross generations to determine the presence of the locus ofinterest. The last backcross generation is usually selfed to give purebreeding progeny for the gene(s) being transferred, although a backcrossconversion with a stably introgressed trait may also be maintained byfurther backcrossing to the recurrent parent with selection for theconverted trait.

Along with selection for the trait of interest, progeny are selected forthe phenotype and/or genotype of the recurrent parent. Whileoccasionally additional polynucleotide sequences or genes may betransferred along with the backcross conversion, the backcrossconversion variety “fits into the same hybrid combination as therecurrent parent inbred variety and contributes the effect of theadditional locus added through the backcross.” Poehlman et al (1995)Breeding Field Crop, 4th Ed., Iowa State University Press, Ames, Iowa,pp. 132-155 and 321-344. When one or more traits are introgressed intothe variety a difference in quantitative agronomic traits, such as yieldor dry down, between the variety and an introgressed version of thevariety in some environments may occur. For example, the introgressedversion may provide a net yield increase in environments where the traitprovides a benefit, such as when a variety with an introgressed traitfor insect resistance is grown in an environment where insect pressureexists, or when a variety with herbicide tolerance is grown in anenvironment where herbicide is used.

One process for adding or modifying a trait or locus in maize varietyPH4D3N comprises crossing PH4D3N plants grown from PH4D3N seed withplants of another maize variety that comprise the desired trait orlocus, selecting F1 progeny plants that comprise the desired trait orlocus to produce selected F1 progeny plants, crossing the selectedprogeny plants with the PH4D3N plants to produce backcross progenyplants, selecting for backcross progeny plants that have the desiredtrait or locus and the phenotypic characteristics of maize varietyPH4D3N to produce selected backcross progeny plants; and backcrossing toPH4D3N one or more times in succession to produce backcross progenyplants that comprise the trait or locus.

The modified PH4D3N or a plant otherwise derived from PH4D3N may befurther characterized as having all or essentially all of the phenotypiccharacteristics, or essentially all of the morphological andphysiological characteristics of maize variety PH4D3N, such as thoselisted in Table 1 and/or may be characterized by percent identity toPH4D3N as determined by molecular markers, such as SSR markers or SNPmarkers. By essentially all of the phenotypic or morphological andphysiological characteristics, it is meant that all of thecharacteristics of a plant are recovered that are otherwise present whencompared in the same environment, other than an occasional variant traitthat might arise during backcrossing or direct introduction of atransgene. Such traits may be determined, for example, relative to thetraits listed in Table 1 as determined at the 5% significance level whengrown under the same environmental conditions.

In addition, the above process and other similar processes describedherein may be used to produce F1 hybrid maize seed by adding a step atthe end of the process that comprises crossing PH4D3N with the locusconversion with a different maize plant and harvesting the resultant F1hybrid maize seed.

Traits are also used by those of ordinary skill in the art tocharacterize progeny. Traits are commonly evaluated at a significancelevel, such as a 1%, 5% or 10% significance level, when measured inplants grown in the same environmental conditions.

Male Sterility and Hybrid Seed Production

Hybrid seed production requires elimination or inactivation of pollenproduced by the female inbred parent. Incomplete removal or inactivationof the pollen provides the potential for self-pollination. A reliablemethod of controlling male fertility in plants offers the opportunityfor improved seed production.

PH4D3N can be produced in a male-sterile form. There are several ways inwhich a maize plant can be manipulated so that it is male sterile. Theseinclude use of manual or mechanical emasculation (or detasseling), useof one or more genetic factors that confer male sterility, includingcytoplasmic genetic and/or nuclear genetic male sterility, use ofgametocides and the like. A male sterile designated PH4D3N may includeone or more genetic factors, which result in cytoplasmic genetic and/ornuclear genetic male sterility. All of such embodiments are within thescope of the present claims. The male sterility may be either partial orcomplete male sterility.

Hybrid maize seed is often produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twomaize inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Provided that there issufficient isolation from sources of foreign maize pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious detasseling process can be avoided by using cytoplasmicmale-sterile (CMS) inbreds. Plants of a CMS inbred are male sterile as aresult of genetic factors in the cytoplasm, as opposed to the nucleus,and so nuclear linked genes are not transferred during backcrossing.Thus, this characteristic is inherited exclusively through the femaleparent in maize plants, since only the female provides cytoplasm to thefertilized seed. CMS plants are fertilized with pollen from anotherinbred that is not male-sterile. Pollen from the second inbred may ormay not contribute genes that make the hybrid plants male-fertile, andeither option may be preferred depending on the intended use of thehybrid. The same hybrid seed, a portion produced from detasseled fertilemaize and a portion produced using the CMS system, can be blended toinsure that adequate pollen loads are available for fertilization whenthe hybrid plants are grown. CMS systems have been successfully usedsince the 1950's and the male sterility trait is routinely backcrossedinto inbred varieties.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219, and chromosomal translocations as described inU.S. Pat. Nos. 3,861,709 and 3,710,511. In addition to these methodsU.S. Pat. No. 5,432,068 describes a system of nuclear male sterilitywhich includes: identifying a gene which is needed for male fertility;silencing this native gene which is needed for male fertility; removingthe native promoter from the essential male fertility gene and replacingit with an inducible promoter; inserting this genetically engineeredgene back into the plant; and thus creating a plant that is male sterilebecause the inducible promoter is not “on” resulting in the malefertility gene not being transcribed. Fertility is restored by inducing,or turning “on”, the promoter, which in turn allows the gene thatconfers male fertility to be transcribed.

These, and the other methods of conferring genetic male sterility in theart, each possess their own benefits and drawbacks. Some other methodsuse a variety of approaches such as delivering into the plant a geneencoding a cytotoxic substance associated with a male tissue specificpromoter or an antisense system in which a gene needed for fertility isidentified and an antisense to that gene is inserted in the plant (seeEP 89/3010153.8 publication no. 329,308 and PCT applicationPCT/CA90/00037 published as WO 90/08828).

Another system for controlling male sterility makes use of gametocides.Gametocides are not a genetic system, but rather a topical applicationof chemicals. These chemicals affect cells that are needed for malefertility. The application of these chemicals affects fertility in theplants only for the growing season in which the gametocide is applied(see U.S. Pat. No. 4,936,904). Application of the gametocide, timing ofthe application and genotype specificity often limit the usefulness ofthe approach and it is not appropriate in all situations.

Incomplete control over male fertility may result in self-pollinatedseed being unintentionally harvested and packaged with hybrid seed. Thiswould typically be only female parent seed, because the male plant isgrown in rows that are typically destroyed prior to seed development.Once the seed from the hybrid bag is planted, it is possible to identifyand select these self-pollinated plants. These self-pollinated plantswill be one of the inbred varieties used to produce the hybrid. Thoughthe possibility of PH4D3N being included in a hybrid seed bag exists,the occurrence is very low because much care is taken by seed companiesto avoid such inclusions. It is worth noting that hybrid seed is sold togrowers for the production of grain or forage and not for breeding orseed production. These self-pollinated plants can be identified andselected by one skilled in the art due to their less vigorous appearancefor vegetative and/or reproductive characteristics, including shorterplant height, small ear size, ear and kernel shape, or othercharacteristics.

Identification of these self-pollinated varieties can also beaccomplished through molecular marker analyses. Through thesetechnologies, the homozygosity of the self-pollinated variety can beverified by analyzing allelic composition at various loci along thegenome. Those methods allow for rapid identification of the plantsdisclosed herein.

Transformation

Transgenes and transformation methods facilitate engineering of thegenome of plants to contain and express heterologous genetic elements,such as foreign genetic elements, or additional copies of endogenouselements, or modified versions of native or endogenous genetic elementsin order to alter at least one trait of a plant in a specific manner.Any sequences, such as DNA, whether from a different species or from thesame species, which have been stably inserted into a genome usingtransformation are referred to herein collectively as “transgenes”and/or “transgenic events”. Transgenes can be moved from one genome toanother using breeding techniques which may include crossing,backcrossing or double haploid production. In some embodiments, atransformed variant of PH4D3N may comprise at least one transgene butcould contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or no more than15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2. Transformed versionsof the claimed maize variety PH4D3N as well as hybrid combinationscontaining and inheriting the transgene thereof are provided. F1 hybridseed are provided which are produced by crossing a different maize plantwith maize variety PH4D3N comprising a transgene introduced into maizevariety PH4D3N by backcrossing or genetic transformation and isinherited by the F1 hybrid seed.

Numerous methods for plant transformation have been developed, includingbiological and physical plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, Glick,B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88 and Armstrong, “The First Decade of Maize Transformation: A Reviewand Future Perspective” (Maydica 44:101-109, 1999). In addition,expression vectors and in vitro culture methods for plant cell or tissuetransformation and regeneration of plants are available. See, forexample, Gruber et al., “Vectors for Plant Transformation” in Methods inPlant Molecular Biology and Biotechnology, Glick, B. R. and Thompson, J.E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

In general, methods to transform, modify, edit or alter plant endogenousgenomic DNA include altering the plant native DNA sequence or apre-existing transgenic sequence including regulatory elements, codingand non-coding sequences. These methods can be used, for example, totarget nucleic acids to pre-engineered target recognition sequences inthe genome. Such pre-engineered target sequences may be introduced bygenome editing or modification. As an example, a genetically modifiedplant variety is generated using “custom” or engineered endonucleasessuch as meganucleases produced to modify plant genomes (see e.g., WO2009/114321; Gao et al. (2010) Plant Journal 1:176-187). Anothersite-directed engineering method is through the use of zinc fingerdomain recognition coupled with the restriction properties ofrestriction enzyme. See e.g., Urnov, et al., (2010) Nat Rev Genet.11(9):636-46; Shukla, et al., (2009) Nature 459 (7245):437-41. Atranscription activator-like (TAL) effector-DNA modifying enzyme (TALEor TALEN) is also used to engineer changes in plant genome. See e.g.,US20110145940, Cermak et al., (2011) Nucleic Acids Res. 39(12) and Bochet al., (2009), Science 326(5959): 1509-12. Site-specific modificationof plant genomes can also be performed using the bacterial type IICRISPR (clustered regularly interspaced short palindromic repeats)/Cas(CRISPR-associated) system. See e.g., Belhaj et al., (2013), PlantMethods 9: 39; The Cas9/guide RNA-based system allows targeted cleavageof genomic DNA guided by a customizable small noncoding RNA in plants(see e.g., WO 2015026883A1).

Plant transformation methods may involve the construction of anexpression vector. Such a vector comprises a DNA sequence that containsa gene under the control of or operatively linked to a regulatoryelement, for example a promoter. The vector may contain one or moregenes and one or more regulatory elements.

A transgenic event which has been stably engineered into the germ cellline of a particular maize plant using transformation techniques, couldbe moved into the germ cell line of another variety using traditionalbreeding techniques that are well known in the plant breeding arts. Forexample, a backcrossing approach is commonly used to move a transgenicevent from a transformed maize plant to another variety, and theresulting progeny would then comprise the transgenic event(s). Also, ifan inbred variety was used for the transformation then the transgenicplants could be crossed to a different inbred in order to produce atransgenic hybrid maize plant.

Various genetic elements can be introduced into the plant genome usingtransformation. These elements include, but are not limited to genes;coding sequences; inducible, constitutive, and tissue specificpromoters; enhancing sequences; and signal and targeting sequences. Forexample, see the traits, genes and transformation methods listed in U.S.Pat. Nos. 6,118,055 and 6,284,953. In addition, transformability of avariety can be increased by introgressing the trait of hightransformability from another variety known to have hightransformability, such as Hi-II. See US Patent PublicationUS2004/0016030.

With transgenic or genetically modified plants, a foreign protein can beproduced in commercial quantities. Thus, techniques for the selectionand propagation of transformed plants, which are well understood in theart, yield a plurality of transgenic or genetically modified plants thatare harvested in a conventional manner, and a foreign protein then canbe extracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114: 92-6(1981).

Transgenic events can be mapped by one of ordinary skill in the art andsuch techniques are well known to those of ordinary skill in the art.For exemplary methodologies in this regard, see for example, Glick andThompson, Methods in Plant Molecular Biology and Biotechnology 269-284(CRC Press, Boca Raton, 1993).

Plants can be genetically engineered or modified to express variousphenotypes of agronomic interest. Through the transformation ormodification of maize the expression of genes can be altered to enhancedisease resistance, insect resistance, herbicide tolerance, agronomictraits, grain quality and other traits. Transformation can also be usedto insert DNA sequences which control or help control male-sterility.DNA sequences native to maize as well as non-native DNA sequences can betransformed into maize and used to alter levels of native or non-nativeproteins. Various promoters, targeting sequences, enhancing sequences,and other DNA sequences can be inserted into the maize genome for thepurpose of altering the expression of proteins. Reduction of theactivity of specific genes (also known as gene silencing, or genesuppression) is desirable for several aspects of genetic engineering inplants.

Many techniques for gene silencing are well known to one of skill in theart, including but not limited to knock-outs (such as by insertion of atransposable element such as mu (Vicki Chandler, The Maize Handbook Ch.118 (Springer-Verlag 1994) or other genetic elements such as a FRT, Loxor other site specific integration site, antisense technology (see,e.g., Sheehy et al. (1988) PNAS USA 85:8805-8809; and U.S. Pat. Nos.5,107,065; 5,453,566; and 5,759,829); co-suppression (e.g., Taylor(1997) Plant Cell 9:1245; Jorgensen (1990) Trends Biotech.8(12):340-344; Flavell (1994) PNAS USA 91:3490-3496; Finnegan et al.(1994) Bio/Technology 12: 883-888; and Neuhuber et al. (1994) Mol. Gen.Genet. 244:230-241); RNA interference (Napoli et al. (1990) Plant Cell2:279-289; U.S. Pat. No. 5,034,323; Sharp (1999) Genes Dev. 13:139-141;Zamore et al. (2000) Cell 101:25-33; and Montgomery et al. (1998) PNASUSA 95:15502-15507), virus-induced gene silencing (Burton, et al. (2000)Plant Cell 12:691-705; and Baulcombe (1999) Curr. Op. Plant Bio.2:109-113); target-RNA-specific ribozymes (Haseloff et al. (1988) Nature334: 585-591); hairpin structures (Smith et al. (2000) Nature407:319-320; WO 99/53050; and WO 98/53083); MicroRNA (Aukerman and Sakai(2003) Plant Cell 15:2730-2741); ribozymes (Steinecke et al. (1992) EMBOJ. 11:1525; and Perriman et al. (1993) Antisense Res. Dev. 3:253);oligonucleotide mediated targeted modification (e.g., WO 03/076574 andWO 99/25853); Zn-finger targeted molecules (e.g., WO 01/52620; WO03/048345; and WO 00/42219); and other methods or combinations of theabove methods known to those of skill in the art.

Exemplary nucleotide sequences that may be altered by geneticengineering include, but are not limited to, those categorized below.

1. Transgenes that Confer Resistance to Insects or Disease and thatEncode:

(A) Plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266: 789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262: 1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78: 1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae), McDowell and Woffenden, (2003) Trends Biotechnol.21(4): 178-83 and Toyoda et al., (2002) Transgenic Res. 11(6):567-82. Aplant resistant to a disease is one that is more resistant to a pathogenas compared to the wild type plant.

(B) A Bacillus thuringiensis protein, a derivative thereof or asynthetic polypeptide modeled thereon. See, for example, Geiser et al.,Gene 48: 109 (1986), who disclose the cloning and nucleotide sequence ofa Bt delta-endotoxin gene. Moreover, DNA molecules encodingdelta-endotoxin genes can be purchased from American Type CultureCollection (Rockville, Md.), for example, under ATCC Accession Nos.40098, 67136, 31995 and 31998. Other non-limiting examples of Bacillusthuringiensis transgenes being genetically engineered are given in thefollowing patents and patent publications: U.S. Pat. Nos. 5,188,960;5,689,052; 5,880,275; 5,986,177; 7,105,332; 7,208,474, 7,329,736;7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,605,304, 7,696,412,7,629,504, 7,772,465, 7,790,846, 7,858,849, WO 91/14778; WO 99/31248; WO01/12731; WO 99/24581; and WO 97/40162.

(C) An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344: 458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

(D) An insect-specific peptide which, upon expression, disrupts thephysiology of the affected pest. For example, see Regan, J. Biol. Chem.269: 9 (1994) (expression cloning yields DNA coding for insect diuretichormone receptor); Pratt et al., Biochem. Biophys. Res. Comm. 163: 1243(1989) (an allostatin is identified in Diploptera puntata);Chattopadhyay et al. (2004) Critical Reviews in Microbiology 30 (1):33-54 2004; Zjawiony (2004) J Nat Prod 67 (2): 300-310; Carlini andGrossi-de-Sa (2002) Toxicon, 40 (11): 1515-1539; Ussuf et al. (2001)Curr Sci. 80 (7): 847-853; and Vasconcelos and Oliveira (2004) Toxicon44 (4): 385-403. See also U.S. Pat. No. 5,266,317 disclosing genesencoding insect-specific toxins.

(E) An enzyme responsible for a hyperaccumulation of a monoterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

(F) An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23: 691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21: 673 (1993), who provide the nucleotide sequenceof the parsley ubi4-2 polyubiquitin gene, and U.S. Pat. Nos. 6,563,020;7,145,060 and 7,087,810.

(G) A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24: 757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104: 1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

(H) A hydrophobic moment peptide. See PCT application WO 95/16776 andU.S. Pat. No. 5,580,852 disclosure of peptide derivatives of Tachyplesinwhich inhibit fungal plant pathogens) and PCT application WO 95/18855and U.S. Pat. No. 5,607,914 (teaches synthetic antimicrobial peptidesthat confer disease resistance).

(I) A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure by Jaynes et al., Plant Sci. 89: 43 (1993),of heterologous expression of a cecropin-beta lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

(J) A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28: 451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

(K) An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, SEVENTH INT'L SYMPOSIUM ON MOLECULARPLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

(L) A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366: 469 (1993), which shows that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

(M) A developmental-arrestive protein produced in nature by a pathogenor a parasite. Thus, fungal endo alpha-1,4-D-polygalacturonasesfacilitate fungal colonization and plant nutrient release bysolubilizing plant cell wall homo-alpha-1,4-D-galacturonase. See Lamb etal., Bio/Technology 10: 1436 (1992). The cloning and characterization ofa gene which encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2: 367 (1992).

(N) A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10: 305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, S., Current Biology, 5(2)(1995), Pieterse and Van Loon (2004) Curr. Opin. Plant Bio. 7(4):456-64and Somssich (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, Pl. Physiol.101:709-712, (1993) and Parijs et al., Planta 183:258-264, (1991) andBushnell et al., Can. J. of Plant Path. 20(2):137-149 (1998). Also seeU.S. application Ser. Nos. 09/950,933; 11/619,645; 11/657,710;11/748,994; 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946.

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255;5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.

(R) Cystatin and cysteine proteinase inhibitors. See U.S. Pat. No.7,205,453.

(S) Defensin genes. See WO03000863 and U.S. Pat. Nos. 6,911,577;6,855,865; 6,777,592 and 7,238,781.

(T) Genes conferring resistance to nematodes. See e.g. PCT ApplicationWO96/30517; PCT Application WO93/19181, WO 03/033651 and Urwin et al.,Planta 204:472-479 (1998), Williamson (1999) Curr Opin Plant Bio.2(4):327-31; and U.S. Pat. Nos. 6,284,948 and 7,301,069.

(U) Genes that confer resistance to Phytophthora Root Rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.See, for example, Shoemaker et al, Phytophthora Root Rot Resistance GeneMapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

(V) Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035.

(W) Genes that confer resistance to Colletotrichum, such as described inUS Patent Publication No. US20090035765. This includes the Rcg locusthat may be utilized as a single locus conversion.

2. Transgenes that Confer Tolerance to a Herbicide, for example:

(A) A herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant acetolactate synthase (ALS) and acetohydroxyacid synthase(AHAS) enzyme as described, for example, in U.S. Pat. Nos. 5,605,011;5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373;5,331,107; 5,928,937; and 5,378,824; US Patent Publication No.20070214515, and international publication WO 96/33270.

(B) Glyphosate (tolerance imparted by aroA genes and mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and) and other phosphonocompounds such as glufosinate (phosphinothricin acetyl transferase (PAT)and Streptomyces hygroscopicus phosphinothricin acetyl transferase (bar)genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones(ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No.4,940,835, which discloses the nucleotide sequence of a form of EPSPSwhich can confer glyphosate tolerance. U.S. Pat. No. 5,627,061 alsodescribes genes encoding EPSPS enzymes. See also U.S. Pat. Nos.6,566,587; 6,338,961; 6,248,876; 6,040,497; 5,804,425; 5,633,435;5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775;6,225,114; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448;5,510,471; RE. 36,449; RE 37,287; and 5,491,288; and internationalpublications EP1173580; WO 01/66704; EP1173581 and EP1173582.

Glyphosate tolerance is also imparted to plants that express a gene thatencodes a glyphosate oxido-reductase enzyme as described more fully inU.S. Pat. Nos. 5,776,760 and 5,463,175. In addition glyphosate tolerancecan be imparted to plants by the over expression of genes encodingglyphosate N-acetyltransferase. See, for example, US2004/0082770;US2005/0246798; and US2008/0234130. A DNA molecule encoding a mutantaroA gene can be obtained under ATCC accession No. 39256, and thenucleotide sequence of the mutant gene is disclosed in U.S. Pat. No.4,769,061. EP Patent Application No. 0333033 and U.S. Pat. No. 4,975,374disclose nucleotide sequences of glutamine synthetase genes which confertolerance to herbicides such as L-phosphinothricin. The nucleotidesequence of a phosphinothricin-acetyl-transferase gene is provided in EPPatent Nos. 0242246 and 0242236. See also, U.S. Pat. Nos. 5,969,213;5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477;5,646,024; 6,177,616 B1; and 5,879,903. Exemplary genes conferringresistance to phenoxy propionic acids, cyclohexanediones andcyclohexones, such as sethoxydim and haloxyfop, are the Acc1-S1, Acc1-S2and Acc1-S3 genes.

(C) A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene) such as bromoxynil.Przibilla et al., Plant Cell 3: 169 (1991), describe the transformationof Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotidesequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648to Stalker, and DNA molecules containing these genes are available underATCC Accession Nos. 53435, 67441 and 67442.

(D) Other genes that confer tolerance to herbicides include: a geneencoding a chimeric protein of rat cytochrome P4507A1 and yeastNADPH-cytochrome P450 oxidoreductase, genes for glutathione reductaseand superoxide dismutase, and genes for various phosphotransferases.

(E) A herbicide that inhibits protoporphyrinogen oxidase (protox or PPO)is necessary for the production of chlorophyll, which is necessary forall plant survival. The protox enzyme serves as the target for a varietyof herbicidal compounds. PPO-inbibitor herbicides can inhibit growth ofall the different species of plants present, causing their totaldestruction. The development of plants containing altered protoxactivity which are tolerant to these herbicides are described, forexample, in U.S. Pat. Nos. 6,288,306 B1; 6,282,837 B1; and 5,767,373;and international patent publication WO 01/12825.

(F) Dicamba (3,6-dichloro-2-methoxybenzoic acid) is an organochloridederivative of benzoic acid which functions by increasing plant growthrate such that the plant dies.

3. Transgenes that Confer or Contribute to an Altered GrainCharacteristic, such as:

(A) Altered fatty acids, for example, by

(i) Down-regulation of stearoyl-ACP desaturase to increase stearic acidcontent of the plant. See Knultzon et al., Proc. Natl. Acad. Sci. USA89: 2624 (1992) and WO99/64579 (Genes for Desaturases to Alter LipidProfiles in Corn),

(ii) Elevating oleic acid via FAD-2 gene modification and/or decreasinglinolenic acid via FAD-3 gene modification (see U.S. Pat. Nos.6,063,947; 6,323,392; 6,372,965 and WO 93/11245),

(iii) Altering linolenic or linoleic acid content, such as in WO01/12800,

(iv) Altering LEC1, AGP, Dek1, Superal1, mi1ps, various Ipa genes suchas Ipa1, Ipa3, hpt or hggt. For example, see WO 02/42424, WO 98/22604,WO 03/011015, WO02/057439, WO03/011015, U.S. Pat. Nos. 6,423,886,6,197,561, 6,825,397, and U.S. Application Serial Nos. US2003/0079247,US2003/0204870, and Rivera-Madrid, R. et al. Proc. Natl. Acad. Sci.92:5620-5624 (1995).

B) Altered phosphate content, for example, by the

(i) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see Van Hartingsveldt et al., Gene 127: 87 (1993), for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(ii) Modulating a gene that reduces phytate content. In maize, this, forexample, could be accomplished, by cloning and then re-introducing DNAassociated with one or more of the alleles, such as the LPA alleles,identified in maize mutants characterized by low levels of phytic acid,such as in WO 05/113778 and/or by altering inositol kinase activity asin WO 02/059324, US2003/0009011, WO 03/027243, US2003/0079247, WO99/05298, U.S. Pat. Nos. 6,197,561, 6,291,224, 6,391,348, WO2002/059324,US2003/0079247, Wo98/45448, WO99/55882, WO01/04147.

(C) Altered carbohydrates affected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or, a genealtering thioredoxin such as NTR and/or TRX (see. (See U.S. Pat. No.6,531,648) and/or a gamma zein knock out or mutant such as cs27 orTUSC27 or en27 (See U.S. Pat. No. 6,858,778 and US2005/0160488,US2005/0204418). See Shiroza et al., J. Bacteriol. 170: 810 (1988)(nucleotide sequence of Streptococcus mutans fructosyltransferase gene),Steinmetz et al., Mol. Gen. Genet. 200: 220 (1985) (nucleotide sequenceof Bacillus subtilis levansucrase gene), Pen et al., Bio/Technology 10:292 (1992) (production of transgenic plants that express Bacilluslicheniformis alpha-amylase), Elliot et al., Plant Molec. Biol. 21: 515(1993) (nucleotide sequences of tomato invertase genes), Søgaard et al.,J. Biol. Chem. 268: 22480 (1993) (site-directed mutagenesis of barleyalpha-amylase gene), and Fisher et al., Plant Physiol. 102: 1045 (1993)(maize endosperm starch branching enzyme II), WO 99/10498 (improveddigestibility and/or starch extraction through modification ofUDP-D-xylose 4-epimerase, Fragile 1 and 2, Ref1, HCHL, C4H), U.S. Pat.No. 6,232,529 (method of producing high oil seed by modification ofstarch levels (AGP)). The fatty acid modification genes mentioned hereinmay also be used to affect starch content and/or composition through theinterrelationship of the starch and oil pathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see U.S. Pat. No. 6,787,683,US2004/0034886 and WO 00/68393 involving the manipulation of antioxidantlevels, and WO 03/082899 through alteration of a homogentisate geranylgeranyl transferase (hggt).

(E) Altered essential seed amino acids. For example, see U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO99/40209 (alteration of amino acid compositions inseeds), WO99/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO98/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO98/56935 (plant amino acid biosyntheticenzymes), WO98/45458 (engineered seed protein having higher percentageof essential amino acids), WO98/42831 (increased lysine), U.S. Pat. No.5,633,436 (increasing sulfur amino acid content), U.S. Pat. No.5,559,223 (synthetic storage proteins with defined structure containingprogrammable levels of essential amino acids for improvement of thenutritional value of plants), WO96/01905 (increased threonine),WO95/15392 (increased lysine), US2003/0163838, US2003/0150014,US2004/0068767, U.S. Pat. No. 6,803,498, WO01/79516.

4. Genes that Control Male-sterility

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is needed for male fertility; silencing this native genewhich is needed for male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT (WO 01/29237).

(B) Introduction of stamen-specific promoters (WO 92/13956, WO92/13957).

(C) Introduction of the barnase and the barstar gene (Paul et al. PlantMol. Biol. 19:611-622, 1992).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014; and 6,265,640.

5. Genes that create a site for site specific DNA integration. Thisincludes the introduction of FRT sites that may be used in the FLP/FRTsystem and/or Lox sites that may be used in the Cre/Loxp system. Forexample, see Lyznik, et al., Site-Specific Recombination for GeneticEngineering in Plants, Plant Cell Rep (2003) 21:925-932 and WO 99/25821.Other systems that may be used include the Gin recombinase of phage Mu(Maeser et al., 1991; Vicki Chandler, The Maize Handbook Ch. 118(Springer-Verlag 1994), the Pin recombinase of E. coli (Enomoto et al.,1983), and the R/RS system of the pSR1 plasmid (Araki et al., 1992).

6. Genes that affect abiotic stress resistance (including but notlimited to flowering, ear and seed development, enhancement of nitrogenutilization efficiency, altered nitrogen responsiveness, droughtresistance or tolerance, cold resistance or tolerance, and saltresistance or tolerance) and increased yield under stress. For example,see: WO 00/73475 where water use efficiency is altered throughalteration of malate; U.S. Pat. Nos. 5,892,009; 5,965,705; 5,929,305;5,891,859; 6,417,428; 6,664,446; 6,706,866; 6,717,034; 6,801,104;WO2000060089; WO2001026459; WO2001035725; WO2001034726; WO2001035727;WO2001036444; WO2001036597; WO2001036598; WO2002015675; WO2002017430;WO2002077185; WO2002079403; WO2003013227; WO2003013228; WO2003014327;WO2004031349; WO2004076638; WO9809521; and WO9938977 describing genes,including CBF genes and transcription factors effective in mitigatingthe negative effects of freezing, high salinity, and drought on plants,as well as conferring other positive effects on plant phenotype;US2004/0148654 and WO01/36596 where abscisic acid is altered in plantsresulting in improved plant phenotype such as increased yield and/orincreased tolerance to abiotic stress; WO2000/006341, WO04/090143, U.S.application Ser. Nos. 10/817,483 and 09/545,334 where cytokininexpression is modified resulting in plants with increased stresstolerance, such as drought tolerance, and/or increased yield. Also seeWO0202776, WO2003052063, JP2002281975, U.S. Pat. No. 6,084,153,WO0164898, U.S. Pat. Nos. 6,177,275 and 6,107,547 (enhancement ofnitrogen utilization and altered nitrogen responsiveness). For ethylenealteration, see US20040128719, US20030166197 and WO200032761. For planttranscription factors or transcriptional regulators of abiotic stress,see e.g. US20040098764 or US20040078852.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g.WO97/49811 (LHY), WO98/56918 (ESD4), WO97/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO96/14414 (CON),WO96/38560, WO01/21822 (VRN1), WO00/44918 (VRN2), WO99/49064 (GI),WO00/46358 (FRI), WO97/29123, U.S. Pat. Nos. 6,794,560, 6,307,126 (GAI),WO99/09174 (D8 and Rht), WO2004076638 and WO2004031349 (transcriptionfactors).

Using PH4D3N to Develop Another Maize Plant

Maize varieties such as PH4D3N are typically developed for use in theproduction of hybrid maize varieties. However, varieties such as PH4D3Nalso provide a source of breeding material that may be used to developnew maize inbred varieties. Plant breeding techniques known in the artand used in a maize plant breeding program include, but are not limitedto, recurrent selection, mass selection, bulk selection, mass selection,backcrossing, pedigree breeding, open pollination breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection, making double haploids, and transformation. Oftencombinations of these techniques are used. The development of maizehybrids in a maize plant breeding program requires, in general, thedevelopment of homozygous inbred varieties, the crossing of thesevarieties, and the evaluation of the crosses. There are many analyticalmethods available to evaluate the result of a cross. The oldest and mosttraditional method of analysis is the observation of phenotypic traitsbut genotypic analysis may also be used.

Methods for producing a maize plant by crossing a first parent maizeplant with a second parent maize plant wherein either the first orsecond parent maize plant is a maize plant of the variety PH4D3N areprovided. The other parent may be any other maize plant, such as anotherinbred variety or a plant that is part of a synthetic or naturalpopulation. Any such methods may be used with the maize variety PH4D3Nsuch as selfing, sibbing, backcrosses, mass selection, pedigreebreeding, bulk selection, hybrid production, crosses to populations, andthe like. These methods are well known in the art and some of the morecommonly used breeding methods are described below.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asPH4D3N and one other inbred variety having one or more desirablecharacteristics that is lacking or which complements PH4D3N. If the twooriginal parents do not provide all the desired characteristics, othersources can be included in the breeding population. In the pedigreemethod, superior plants are selfed and selected in successive filialgenerations. In the succeeding filial generations the heterozygouscondition gives way to homogeneous varieties as a result ofself-pollination and selection. Typically in the pedigree method ofbreeding, five or more successive filial generations of selfing andselection is practiced: F1→F2; F2→F3; F3→F4; F4→F5, etc. After asufficient amount of inbreeding, successive filial generations willserve to increase seed of the developed inbred. Preferably, the inbredvariety comprises homozygous alleles at about 95% or more of its loci.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. PH4D3N is suitable for use in arecurrent selection program. The method entails individual plants crosspollinating with each other to form progeny. The progeny are grown andthe superior progeny selected by any number of selection methods, whichinclude individual plant, half-sib progeny, full-sib progeny, selfedprogeny and topcrossing. The selected progeny are cross pollinated witheach other to form progeny for another population. This population isplanted and again superior plants are selected to cross pollinate witheach other. Recurrent selection is a cyclical process and can berepeated as many times as desired. The objective of recurrent selectionis to improve the traits of a population. The improved population canthen be used as a source of breeding material to obtain inbred varietiesto be used in hybrids or used as parents for a synthetic cultivar. Asynthetic cultivar is the resultant progeny formed by the intercrossingof several selected inbreds.

PH4D3N is suitable for use in mass selection. Mass selection is a usefultechnique when used in conjunction with molecular marker enhancedselection. In mass selection seeds from individuals are selected basedon phenotype and/or genotype. These selected seeds are then bulked andused to grow the next generation. Bulk selection requires growing apopulation of plants in a bulk plot, allowing the plants toself-pollinate, harvesting the seed in bulk and then using a sample ofthe seed harvested in bulk to plant the next generation. Instead of selfpollination, directed pollination could be used as part of the breedingprogram.

Mutation Breeding

Mutation breeding is one of many methods that could be used to introducenew traits into PH4D3N. PH4D3N is suitable for use in a mutationbreeding program. Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means including temperature, long-term seed storage, tissueculture conditions, radiation; such as X-rays, Gamma rays (e.g. cobalt60 or cesium 137), neutrons, (product of nuclear fission by uranium 235in an atomic reactor), Beta radiation (emitted from radioisotopes suchas phosphorus 32 or carbon 14), or ultraviolet radiation (preferablyfrom 2500 to 2900 nm), or chemical mutagens (such as base analogues(5-bromo-uracil), related compounds (8-ethoxy caffeine), antibiotics(streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards,epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones),azide, hydroxylamine, nitrous acid, or acridines. Once a desired traitis observed through mutagenesis the trait may then be incorporated intoexisting germplasm by traditional breeding techniques, such asbackcrossing. In addition, mutations created in other varieties may beused to produce a backcross conversion of PH4D3N that comprises suchmutation.

Production of Double Haploids

The production of double haploids can also be used for the developmentof inbreds in the breeding program. For example, an F1 hybrid for whichPH4D3N is a parent can be used to produce double haploid plants. Doublehaploids are produced by the doubling of a set of chromosomes (1N) froma heterozygous plant to produce a completely homozygous individual. Forexample, see Wan et al., Theoretical and Applied Genetics, 77:889-892,1989 and US Patent Publication No. 2003/0005479. This can beadvantageous because the process omits the generations of selfing neededto obtain a homozygous plant from a heterozygous source.

Haploid induction systems have been developed for various plants toproduce haploid tissues, plants and seeds. The haploid induction systemcan produce haploid plants from any genotype by crossing a selectedvariety (as female) with an inducer variety. Such inducer varieties formaize include Stock 6 (Coe, 1959, Am. Nat. 93:381-382; Sharkar and Coe,1966, Genetics 54:453-464) RWS (available online from the UniversitätHohenheim), KEMS (Deimling, Roeber, and Geiger, 1997, Vortr.Pflanzenzuchtg 38:203-224), KMS and ZMS (Chalyk, Bylich and Chebotar,1994, MNL 68:47; Chalyk and Chebotar, 2000, Plant Breeding 119:363-364),and indeterminate gametophyte (ig) mutation (Kermicle 1969 Science166:1422-1424).

Methods for obtaining haploid plants are also disclosed in Kobayashi, M.et al., Journ. of Heredity 71(1):9-14, 1980, Pollacsek, M., Agronomie(Paris) 12(3):247-251, 1992; Cho-Un-Haing et al., Journ. of Plant Biol.,1996, 39(3):185-188; Verdoodt, L., et al., February 1998, 96(2):294-300;Genetic Manipulation in Plant Breeding, Proceedings InternationalSymposium Organized by EUCARPIA, Sep. 8-13, 1985, Berlin, Germany;Chalyk et al., 1994, Maize Genet Coop. Newsletter 68:47; Chalyk, S. T.,1999, Maize Genet. Coop. Newsletter 73:53-54; Coe, R. H., 1959, Am. Nat.93:381-382; Deimling, S. et al., 1997, Vortr. Pflanzenzuchtg 38:203-204;Kato, A., 1999, J. Hered. 90:276-280; Lashermes, P. et al., 1988, Theor.Appl. Genet. 76:570-572 and 76:405-410; Tyrnov, V. S. et al., 1984,Dokl. Akad. Nauk. SSSR 276:735-738; Zabirova, E. R. et al., 1996,Kukuruza I Sorgo N4, 17-19; Aman, M. A., 1978, Indian J. Genet PlantBreed 38:452-457; Chalyk S. T., 1994, Euphytica 79:13-18; Chase, S. S.,1952, Agron. J. 44:263-267; Coe, E. H., 1959, Am. Nat. 93:381-382; Coe,E. H., and Sarkar, K. R., 1964 J. Hered. 55:231-233; Greenblatt, I. M.and Bock, M., 1967, J. Hered. 58:9-13; Kato, A., 1990, Maize Genet.Coop. Newsletter 65:109-110; Kato, A., 1997, Sex. Plant Reprod.10:96-100; Nanda, D. K. and Chase, S. S., 1966, Crop Sci. 6:213-215;Sarkar, K. R. and Coe, E. H., 1966, Genetics 54:453-464; Sarkar, K. R.and Coe, E. H., 1971, Crop Sci. 11:543-544; Sarkar, K. R. and Sachan J.K. S., 1972, Indian J. Agric. Sci. 42:781-786; Kermicle J. L., 1969,Mehta Yeshwant, M. R., Genetics and Molecular Biology, September 2000,23(3):617-622; Tahir, M. S. et al. Pakistan Journal of Scientific andIndustrial Research, August 2000, 43(4):258-261; Knox, R. E. et al.Plant Breeding, August 2000, 119(4):289-298; U.S. Pat. No. 5,639,951 andUS Patent Publication No. 20020188965.

Thus, certain embodiments include a process for making a homozygousPH4D3N progeny plant substantially similar to PH4D3N by producing orobtaining a seed from the cross of PH4D3N and another maize plant andapplying double haploid methods to the F1 seed or F1 plant or to anysuccessive filial generation. Such methods decrease the number ofgenerations required to produce an inbred with similar genetics orcharacteristics to PH4D3N. See Bernardo, R. and Kahler, A. L., Theor.Appl. Genet. 102:986-992, 2001.

In particular, a process of making seed substantially retaining themolecular marker profile of maize variety PH4D3N is contemplated, suchprocess comprising obtaining or producing F1 hybrid seed for which maizevariety PH4D3N is a parent, inducing double haploids to create progenywithout the occurrence of meiotic segregation, obtaining the molecularmarker profile of maize variety PH4D3N, and selecting progeny thatretain the molecular marker profile of PH4D3N.

Another embodiment is a maize seed derived from inbred maize varietyPH4D3N produced by crossing a plant or plant part of inbred maizevariety PH4D3N with another plant, wherein representative seed of saidinbred maize variety PH4D3N has been deposited and wherein said maizeseed derived from the inbred maize variety PH4D3N has 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% of the same polymorphisms for molecularmarkers as the plant or plant part of inbred maize variety PH4D3N. Thenumber of molecular markers used for the molecular marker profile can befound in the Panzea database which is available online from Panzea. Thetype of molecular marker used in the molecular profile can be but is notlimited to Single Nucleotide Polymorphisms, SNPs. A maize seed derivedfrom inbred maize variety PH4D3N produced by crossing a plant or plantpart of inbred maize variety PH4D3N with another plant, whereinrepresentative seed of said inbred maize variety PH4D3N has beendeposited and wherein said maize seed derived from the inbred maizevariety PH4D3N has essentially the same morphological characteristics asmaize variety PH4D3N when grown in the same environmental conditions.The same environmental conditions may be, but is not limited to aside-by-side comparison. The characteristics can be those listed inTable 1. The comparison can be made using any number of professionallyaccepted experimental designs and statistical analysis.

Use of PH4D3N in Tissue Culture

Methods of tissue culturing cells of PH4D3N and a tissue culture ofPH4D3N is provided. As used herein, the term “tissue culture” includesplant protoplasts, plant cell tissue culture, cultured microspores,plant calli, plant clumps, and the like. In certain embodiments, thetissue culture comprises embryos, protoplasts, meristematic cells,pollen, leaves or anthers derived from immature tissues of pollen,flowers, kernels, ears, cobs, leaves, husks, stalks, roots, root tips,anthers, silk, and the like. As used herein, phrases such as “growingthe seed” or “grown from the seed” include embryo rescue, isolation ofcells from seed for use in tissue culture, as well as traditionalgrowing methods.

Means for preparing and maintaining plant tissue cultures are well knownin the art See, e.g., U.S. Pat. Nos. 5,538,880; 5,550,318, and6,437,224, the latter describing tissue culture of maize, includingtassel/anther culture. A tissue culture comprising organs such astassels or anthers is provided which can be used to produce regeneratedplants. (See, e.g., U.S. Pat. Nos. 5,445,961 and 5,322,789). Thus, incertain embodiments, cells are provided which upon growth anddifferentiation produce maize plants having the genotype and/orphenotypic characteristics of variety PH4D3N.

Seed Treatments and Cleaning

Methods of harvesting the seed of the maize variety PH4D3N as seed forplanting are provided. Embodiments include cleaning the seed, treatingthe seed, and/or conditioning the seed. Cleaning the seed is understoodin the art to include removal of foreign debris such as one or more ofweed seed, chaff, and plant matter, from the seed. Conditioning the seedis understood in the art to include controlling the temperature and rateof dry down of the seed and storing seed in a controlled temperatureenvironment. Seed treatment is the application of a composition to theseed such as a coating or powder. Methods for producing a treated seedinclude the step of applying a composition to the seed or seed surface.Seeds are provided which have on the surface a composition. Biologicalactive components such as bacteria can also be used as a seed treatment.Some examples of compositions are insecticides, fungicides, pesticides,antimicrobials, germination inhibitors, germination promoters,cytokinins, and nutrients.

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases, thereby furtherenhancing the invention described herein. Seed material can be treated,typically surface treated, with a composition comprising combinations ofchemical or biological herbicides, herbicide safeners, insecticides,fungicides, germination inhibitors and enhancers, nutrients, plantgrowth regulators and activators, bactericides, nematicides, avicidesand/or molluscicides. These compounds are typically formulated togetherwith further carriers, surfactants or application-promoting adjuvantscustomarily employed in the art of formulation. The coatings may beapplied by impregnating propagation material with a liquid formulationor by coating with a combined wet or dry formulation. Examples of thevarious types of compounds that may be used as seed treatments areprovided in The Pesticide Manual: A World Compendium, C. D. S. TomlinEd., Published by the British Crop Production Council.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, Bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis),Bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA registrationnumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits in order to enhanceyield. For example, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

INDUSTRIAL APPLICABILITY

Another embodiment is a method of harvesting the grain or plant materialof the F1 plant of variety PH4D3N and using the grain or plant materialin a commodity. Methods of producing a commodity plant product are alsoprovided. Examples of maize grain or plant material as a commodity plantproduct include, but are not limited to, oils, meals, flour, starches,syrups, proteins, cellulose, silage, and sugars. Maize grain is used ashuman food, livestock feed, and as raw material in industry. The fooduses of maize, in addition to human consumption of maize kernels,include both products of dry- and wet-milling industries. The principalproducts of maize dry milling are grits, meal and flour. The maizewet-milling industry can provide maize starch, maize syrups, anddextrose for food use. Maize oil is recovered from maize germ, which isa by-product of both dry- and wet-milling industries. Processing thegrain can include one or more of cleaning to remove foreign material anddebris from the grain, conditioning, such as addition of moisture to thegrain, steeping the grain, wet milling, dry milling and sifting.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry.

Industrial uses of maize include production of ethanol, maize starch inthe wet-milling industry and maize flour in the dry-milling industry.The industrial applications of maize starch and flour are based onfunctional properties, such as viscosity, film formation, adhesiveproperties, and ability to suspend particles. The maize starch and flourhave application in the paper and textile industries. Other industrialuses include applications in adhesives, building materials, foundrybinders, laundry starches, explosives, oil-well muds, and other miningapplications.

Provided are plant parts other than the grain of maize and their use inindustry: for example, methods for making stalks and husks into paperand wallboard and for using cobs for fuel and to make charcoal areprovided.

The seed of maize variety PH4D3N, the plant produced from the seed, thehybrid maize plant produced from the crossing of the variety, hybridseed, and various parts of the hybrid maize plant and transgenicversions of the foregoing, can be utilized for human food, livestockfeed, and as a raw material in industry.

All publications, patents, and patent applications mentioned in thespecification are incorporated by reference herein for the purpose citedto the same extent as if each was specifically and individuallyindicated to be incorporated by reference herein.

The foregoing invention has been described in detail by way ofillustration and example for purposes of clarity and understanding. Asis readily apparent to one skilled in the art, the foregoing are onlysome of the methods and compositions that illustrate the embodiments ofthe foregoing invention. It will be apparent to those of ordinary skillin the art that variations, changes, modifications, and alterations maybe applied to the compositions and/or methods described herein withoutdeparting from the true spirit, concept, and scope of the invention.

As used herein, the terms “comprises,” “comprising,” “includes,”“including,” “has,” “having,” “contains”, “containing,” “characterizedby” or any other variation thereof, are intended to cover anon-exclusive inclusion.

Unless expressly stated to the contrary, “or” is used as an inclusiveterm. For example, a condition A or B is satisfied by any one of thefollowing: A is true (or present) and B is false (or not present), A isfalse (or not present) and B is true (or present), and both A and B aretrue (or present). The indefinite articles “a” and “an” preceding anelement or component are nonrestrictive regarding the number ofinstances (i.e., occurrences) of the element or component. Therefore “a”or “an” should be read to include one or at least one, and the singularword form of the element or component also includes the plural unlessthe number is obviously meant to be singular.

DEPOSITS

Applicant has made a deposit of at least 625 seeds of Maize VarietyPH4D3N with the Provasoli-Guillard National Center for Marine Algae andMicrobiota (NCMA), 60 Bigelow Drive, East Boothbay, Me. 04544, USA, withNCMA Accession No. 202301022 . The seeds deposited with the NCMA on Jan.18, 2023 were obtained from the seed of the variety maintained byPioneer Hi-Bred International, Inc., 7250 NW 62^(nd) Avenue, Johnston,Iowa, 50131 since prior to the filing date of this application. Accessto this seed will be available during the pendency of the application tothe Commissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon issuance of anyclaims in the application, the Applicant will make the deposit availableto the public pursuant to 37 C.F.R. § 1.808. This deposit of the MaizeVariety PH4D3N will be maintained in the NCMA depository, which is apublic depository, for a period of 30 years, or 5 years after the mostrecent request, or for the enforceable life of the patent, whichever islonger, and will be replaced if it becomes nonviable during that period.Additionally, Applicant has or will satisfy all of the requirements of37 C.F.R. §§ 1.801-1.809, including providing an indication of theviability of the sample upon deposit. Applicant has no authority towaive any restrictions imposed by law on the transfer of biologicalmaterial or its transportation in commerce. Applicant does not waive anyinfringement of rights granted under this patent or under the PlantVariety Protection Act (7 USC 2321 et seq.).

Breeding History of PH4D3N

Inbred Maize variety PH4D3N was developed by the following method.PH4D3N is an elite corn inbred line originated from a cross betweeninbred line PH1VY6 and inbred line PH18WY. From this cross, ears wereselected based on genetic analysis predicting disease, insect, andagronomic phenotypic performance. Inbred PH4D3N was developed byproducing a doubled haploid from the F1 plants, selfing and usingpedigree selection amongst the D1 lines, and selfing and bulking fromthe subsequent generations.

Maize variety PH4D3N, being substantially homozygous, can be reproducedby planting seeds of the variety, growing the resulting maize plantsunder self-pollinating or sib-pollinating conditions with adequateisolation, and harvesting the resulting seed using techniques familiarto the agricultural arts.

TABLE 1 VARIETY DESCRIPTION INFORMATION - PH4D3N 1. TYPE: Grain Textureintermediate 2. MATURITY: Days Comparative Relative Maturity (CRM) 85Number of Days to Silking from Planting 65 Number of Days to Sheddingfrom Planting 64 3. PLANT: Value Plant Height (to tassel tip) (cm) 258Ear Height (to base of top ear node) (cm) 90 Internode Anthocyanin ColorIntensity absent or very weak Nodes Anthocyanin Color Intensity absentor very weak Brace Roots Anthocyanin Coloration medium Degree of StemZig-Zag absent or very slight Ratio Height of Insertion of Peduncle 0.35of Upper Ear to Plant Length 4. LEAF: Leaf Width of Blade (cm) 8 LeafLength (score) 0.90 m to 1 m Leaf Tip Shape pointed Leaf Angle (betweenblade and stem) small (6 to 37 degrees) Foliage Intensity of Green Colordark Leaf Sheath Hairiness (scale) 1 1 = none to 6 = peach-like fuzzSheath Anthocyanin Color Intensity weak (observed at first leaf stage)Sheath Anthocyanin Color Intensity absent or very weak (whole plant, earinsertion level) Leaf Limb Anthocyanin Color Intensity absent or veryweak (of entire plant) 5. TASSEL: Number of Primary Lateral Branches few(4 to 10) Number of Secondary Branches 0 to 3 branches Tassel AngleBetween Main Axis small to medium and Lateral Branches Length of MainAxis Above 27 Highest Lateral Branch (cm) Length of Main Axis Above 36Lowest Lateral Branch (cm) Tassel Lateral Branch Curvature slightlyrecurved (6 to 37 degrees) Primary Tassel Branch Length long TasselSpikelet Density (score) moderately lax Bar Glume Color Intensity absentAnther Color yellow Glume Color green 6a. EAR: Silk Anthocyanin ColorIntensity absent or very weak Ear Husk Length medium Ear Shank Length(scale) medium Ear Shape (taper) conico-cylindrical Ear Length (cm) 157Ear Diameter (mm) 38 Number of Rows of Grain on Ear 14 7. KERNEL(Dried): Dorsal Side of Grain Color yellow Top of Grain Color yellowKernel shape cuneiform 8. COB: Cob Diameter at mid-point (mm) 21 CobColor Red

What is claimed is:
 1. A seed, plant, plant part, or plant cell ofinbred maize variety PH4D3N, representative seed of the variety havingbeen deposited under NCMA accession No.
 202301022. 2. The plant part ofclaim 1, wherein the plant part is an ovule or pollen.
 3. An F1 hybridmaize seed produced by crossing the plant or plant part of claim 1 witha different maize plant.
 4. An F1 hybrid maize plant or plant partproduced by growing the maize seed of claim 3, wherein the plant partcomprises at least one cell of the F1 hybrid maize plant.
 5. A methodfor producing a second maize plant, the method comprising applying plantbreeding techniques to the F1 plant or plant part of claim 4 to producethe second maize plant.
 6. A method for producing a second maize plantor plant part, the method comprising doubling haploid seed generatedfrom a cross of the plant or plant part of claim 4 with an inducervariety, thereby producing the second maize plant or plant part.
 7. Amethod of making a commodity plant product comprising silage, starch,fat, syrup or protein, the method comprising producing the commodityplant product from the maize plant or plant part of claim
 4. 8. A methodof producing a maize seed derived from the variety PH4D3N, comprising:a) self-pollinating the plant of claim 1 or crossing the plant of claim1 with a second plant to produce progeny seed; and b) growing theprogeny seed to produce a progeny plant and self-pollinating the progenyplant or crossing the progeny plant with a different plant to producemaize seed derived from the variety PH4D3N.
 9. A method for producingnucleic acids, the method comprising isolating nucleic acids from theseed, plant, plant part, or plant cell of claim
 1. 10. A converted seed,plant, plant part or plant cell of inbred maize variety PH4D3N,representative seed of the maize variety PH4D3N having been depositedunder NCMA accession No. 202301022, wherein the converted seed, plant,plant part or plant cell comprises a locus conversion, and wherein theplant or a plant grown from the converted seed, plant part or plant cellcomprises the locus conversion and otherwise comprises the physiologicaland morphological characteristics of maize variety PH4D3N when grownunder the same environmental conditions.
 11. The converted seed, plant,plant part or plant cell of claim 10, wherein the locus conversionconfers a property selected from the group consisting of male sterility,site-specific recombination, abiotic stress tolerance, alteredphosphate, altered antioxidants, altered fatty acids, altered essentialamino acids, altered carbohydrates, herbicide tolerance, insectresistance and disease resistance.
 12. A maize seed produced by crossingthe plant or plant part of claim 10 with a different maize plant.
 13. Ahybrid maize plant or plant part produced by growing the seed of claim12, wherein the plant part comprises at least one cell of the hybridmaize plant.
 14. A method for producing a second maize plant, the methodcomprising applying plant breeding techniques to the plant or plant partof claim 13 to produce the second maize plant.
 15. A method forproducing a second maize plant or plant part, the method comprisingdoubling haploid seed generated from a cross of the plant or plant partof claim 13 with an inducer variety, thereby producing the second maizeplant or plant part.
 16. A method of making a commodity plant productcomprising silage, starch, fat, syrup or protein, the method comprisingproducing the commodity plant product from the maize plant or plant partof claim
 13. 17. A method for producing nucleic acids, the methodcomprising isolating nucleic acids from the seed, plant, plant part, orplant cell of claim
 10. 18. An F1 hybrid seed produced by crossing aplant or plant part of inbred maize variety PH4D3N, representative seedof the variety having been deposited under NCMA accession number202301022 with a different maize plant, wherein inbred maize varietyPH4D3N further comprises a transgene that is inherited by the seed,wherein the transgene was introduced into inbred maize variety PH4D3N bybackcrossing or genetic transformation.
 19. A method of producingprogeny seed, the method comprising self-pollinating a plant grown fromthe seed of claim 18 or crossing a plant grown from the seed of claim 18with a second plant to produce progeny seed.
 20. A method of making acommodity plant product comprising silage, starch, fat, syrup orprotein, the method comprising producing the commodity plant productfrom the maize plant or plant part of claim 18.